Coronavirus proteins and antigens

ABSTRACT

Disclosed herein are embodiments of a method for collecting, extracting or eluting proteins and antigens from cells infected with coronavirus. The coronavirus may be a porcine coronavirus, such as porcine epidemic diarrhea virus (PEDV) or porcine delta coronavirus (PDCoV). Also disclosed are embodiments of a composition comprising the coronavirus proteins and antigens, and embodiments of a method of using such a composition. Applications for the composition include, but are not limited to, use in the preparation of antibodies against the proteins and antigens, use as reference markers for coronavirus proteins, and/or use in an immunogenic composition, such as in a vaccine composition.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/228,898, filed on Aug. 4, 2016, which is a continuation-in-part ofInternational Application No. PCT/US2015/015009, filed on Feb. 9, 2015,which in turn claims the benefit of the earlier filing date of U.S.Provisional Patent Application No. 61/937,419, filed on Feb. 7, 2014.This application also claims the benefit of the earlier filing date ofU.S. Provisional Patent Application No. 62/209,538, filed on Aug. 25,2015, and of International Application No. PCT/US2016/017183, filed onFeb. 9, 2016, which in turn claims the benefit of the earlier filingdates of U.S. Provisional Patent Application Nos. 62/113,976 and62/113,979, both filed on Feb. 9, 2015. Each of these applications isincorporated by reference herein in its entirety.

FIELD

This disclosure relates to the preparation and isolation of coronavirusproteins and antigens, particularly from porcine coronaviruses. Thedisclosure further provides viral proteins and antigens as obtained fromcoronavirus infected cells and compositions comprising the proteins andantigens.

BACKGROUND

Coronaviruses are a family of RNA viruses that infect avians andmammals, including humans and swine. Coronaviruses belong to the familyCoronaviridae, which has four main sub-groupings, known asalphacoronavirus, betacoronavirus, gammacoronavirus anddeltacoronavirus. Human coronaviruses include alphacoronaviruses 229Eand NL63, and betacoronaviruses OC43, HKU1, SARS-CoV (the coronavirusthat causes severe acute respiratory syndrome, or SARS), and MERS-CoV(the coronavirus that causes Middle East Respiratory Syndrome, or MERS).Porcine coronaviruses include alphacoronaviruses, such as transmissiblegastroenteritis virus, porcine respiratory coronavirus, and porcineepidemic diarrhea virus (PEDV); betacoronaviruses, such as porcinehemagglutinating encephalomyelitis virus; and deltacoronavirus, such asporcine deltacoronavirus (PDCoV). Other coronaviruses include, but arenot limited to, bovine coronavirus (BCV), feline coronavirus (FCoV),canine coronavirus (CCoV), avian infectious bronchitis virus (IBV), andturkey coronavirus (TCV). Porcine coronaviruses are important diseasesin swine production. For example, PEDV is a highly infectiouscoronavirus that infects the intestinal system of a pig, typicallycausing diarrhea and dehydration. While adult pigs mostly become sickand lose weight after becoming infected, the virus is often fatal tonewborn piglets. Infected herds can suffer a loss of from 50% to 100% ofthe piglets for a four to five week period. It has been estimated thatbetween June 2013 and March 2014 over 4 million piglets were lost toPEDV in USA.

SUMMARY

This disclosure relates to proteins and antigens from a coronavirus. Thecoronavirus can be any coronavirus currently known, or later discovered.In certain embodiments, the disclosure relates to coronaviruses thatinfect avian or mammals, including, but not limited to, humans, andswine. The coronavirus may be a porcine coronavirus, and may be selectedfrom transmissible gastroenteritis virus, porcine respiratorycoronavirus, PEDV, porcine hemagglutinating encephalomyelitis virus, orPDCoV. Particular embodiments concern proteins and antigens from PEDV,and other particular embodiments concern proteins and antigens fromPDCoV.

Disclosed herein are embodiments of a method of preparing coronavirusproteins and/or antigens, such as PEDV or PDCoV proteins and/orantigens, from cells infected with a coronavirus, such as PEDV or PDCoV.In some embodiments, the proteins and/or antigens are harvested at anearly time point after infection when the majority, or entirety, of theviral proteins and/or antigens remain associated with the infectedcells. In such embodiments, the majority or entirety of coronavirusencoded proteins are either within the infected cells or associated withthe cell membrane of the infected cells. Under such conditions,relatively few, if any, coronavirus particles are present in theextracellular environment outside the cells. In other embodiments, theproteins and/or antigens are harvested at a stage after infection whenat least some of the infected cells have released replicated coronavirusviral particles into the extracellular environment.

Certain embodiments of the disclosed method may include providing apopulation of cultured cells infected with a coronavirus, such as PEDVor PDCoV; isolating and/or separating the coronavirus-infected cellsfrom the culture medium and/or cell-free coronavirus in the medium; andcollecting coronavirus proteins and/or antigens from the isolated cells.Collecting viral proteins and/or antigens may be done by extracting oreluting them from the isolated, infected cells with adetergent-containing solution. In some examples, thedetergent-containing solution comprises an amount of detergent effectiveto extract or elute the proteins and/or antigens, such as 0.5% TritonX-100. In additional embodiments, the collecting, extracting or elutingmay be for up to 24 hours, such as 2 to 15 hours or in some embodiments,from 0.2 to 5 hours. The collecting, extracting or eluting is performedat a temperature suitable for the collecting, extracting or eluting,such as from 0° C. to 25° C., from 2° C. to 10° C., from 2° C. to 6° C.or, in certain examples, 4° C. Optionally, the coronavirus proteinsand/or antigens produced by the method may include coronavirus envelopeproteins, such as PEDV envelope proteins or PDCoV envelope proteins.

Also disclosed herein are isolated coronavirus proteins and/or antigens,such as PEDV or PDCoV proteins and/or antigens, prepared by thedisclosed method. The viral proteins and/or antigens may include one ormore envelope proteins produced by an infected cell. Compositionscomprising the viral proteins and/or antigens are also contemplated.Optionally, the composition may contain one or more adjuvants, and/oradditional excipients, such as a pharmaceutically acceptable carrier.

Embodiments of a method of producing an immune response in a subject,such as a porcine subject, are also disclosed. The method may includeadministering one or more coronavirus proteins and/or antigens, preparedby the disclosed method, to the subject. The one or more coronavirusproteins and/or antigens may comprise one or more proteins and/orantigens from a single coronavirus. Additionally, or alternatively, theone or more coronavirus proteins and/or antigens may comprise proteinsand/or antigens from one or more coronaviruses, such as, for example,PEDV and PDCoV. In certain embodiments, the method comprisesadministering one or more PEDV or PDCoV proteins and/or antigens to aporcine subject. Optionally, producing an immune response may comprisevaccinating the subject. In some embodiments, a composition comprisingthe proteins and/or antigens is administered to the subject. The methodmay also include one or more repeated administration(s) of the proteinsand/or antigens, or the composition, to the same subject, such as 2, 3,4 or more administrations.

Embodiments of a kit comprising the isolated proteins and/or antigens isdisclosed herein. The kit may comprise a composition comprising thecoronavirus proteins and/or antigens. In some embodiments, the kit issuitable for use in a method of producing an immune response in, or amethod of vaccinating, a subject.

The various aspects of the disclosure are contemplated for use inrelation to all coronavirus strains that are antigenically identical, orrelated to, those isolated in North America, Europe and Asia. Therefore,the disclosure may be more generally viewed as based on the protein(s)and/or antigens of any coronavirus isolate, strain or subtype.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Western blot of PEDV proteins from cultures of infectedcells, contacted with two monoclonal antibodies.

FIG. 2 is a Western blot of PEDV proteins from cultures of infectedcells over time, with a mixture of the two monoclonal antibodies used inFIG. 1.

FIG. 3 is a table of data comparing the mortality of piglets fromnon-vaccinated sows and sows vaccinated 3-5 days pre-farrow with anexemplary embodiment of a PEDV vaccine disclosed herein, where thenon-vaccinated and vaccinated sows and litters are kept in the sameroom.

FIG. 4 is a table of data comparing the mortality of piglets fromnon-vaccinated sows and sows vaccinated 3-5 days pre-farrow with anexemplary embodiment of a PEDV vaccine disclosed herein, where thenon-vaccinated and vaccinated sows and litters are kept in separaterooms.

FIG. 5 is a table of data from multiple farms, providing baseline pigletmortality data from non-vaccinated herds.

FIG. 6 is a Western blot illustrating the detection of antibodiesagainst 180-kDa to 350-kDa of PEDV spike protein.

FIG. 7 is a Western blot of three MARC-cell based detergent extracts ofPEDV isolates, illustrating the proteins present in the extracts.

FIG. 8 is a Western blot of the three detergent extracts of FIG. 7 andmixtures of the extracts before and after viral inactivation,illustrating the proteins present before and after inactivation.

FIG. 9 is a table illustrating the PEDV sequence homology between SEQ IDNOS: 1-9.

FIGS. 10A-10C are tables illustrating the PDCoV sequence homologybetween SEQ ID NOS: 18-63.

FIG. 10A is a table illustrating the PDCoV sequence homology concerning15 strains.

FIG. 10B is a table illustrating the PDCoV sequence homology concerninganother 15 strains.

FIG. 10C is a table illustrating the PDCoV sequence homology concerninganother 16 strains.

FIG. 11 is a schematic diagram illustrating the relationship between thePDCoV strains or isolates of SEQ ID NOS: 18-63 in terms of nucleotidesubstitutions per 100 residues.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R. §1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile, created on Aug. 4, 2016, 1.86 MB, which is incorporated byreference herein. In the accompanying sequence listing: SEQ ID NO: 1 isthe nucleotide sequence of North American PEDV strain Colorado 2013(GenBank Accession No. KF272920).

SEQ ID NO: 2 is the nucleotide sequence of North American PEDV strainIowa/18984/2013 (GenBank Accession No. KF804028).

SEQ ID NO: 3 is the nucleotide sequence of North American PEDV strainNorth Carolina USA/NC/2013/35140 (GenBank Accession No. KM975735).

SEQ ID NO: 4 is the nucleotide sequence of North American PEDV strainIndiana12.83/2013 (GenBank Accession No. KJ645635).

SEQ ID NO: 5 is the nucleotide sequence of North American PEDV strainIowa/2013 (GenBank Accession No. KJ645649).

SEQ ID NO: 6 is the nucleotide sequence of North American PEDV strain1251-125-10.

SEQ ID NO: 7 is the nucleotide sequence of Korean PEDV strain SM98(GenBank Accession No. GU937797).

SEQ ID NO: 8 is the nucleotide sequence of Korean attenuated PEDV strainKR-DR13-att (GenBank Accession No. JQ023162).

SEQ ID NO: 9 is the nucleotide sequence of Chinese PEDV strain AH2012(GenBank Accession No. KC210145).

SEQ ID NO: 10 is the deduced amino acid sequence of the S proteinencoded by SEQ ID NO: 1 North American PEDV strain Colorado 2013.

SEQ ID NO: 11 is the deduced amino acid sequence of the S proteinencoded by SEQ ID NO: 2 North American PEDV strain Iowa/18984/2013.

SEQ ID NO: 12 is the deduced amino acid sequence of the S proteinencoded by SEQ ID NO: 3 North American PEDV strain North CarolinaUSA/NC/2013/35140.

SEQ ID NO: 13 is the deduced amino acid sequence of the S proteinencoded by SEQ ID NO: 4 North American PEDV strainUSA/Indiana12.83/2013.

SEQ ID NO: 14 is the deduced amino acid sequence of the S proteinencoded by SEQ ID NO: 5 North American PEDV strain USA/Iowa/2013.

SEQ ID NO: 15 is the deduced amino acid sequence of the S proteinencoded by SEQ ID NO: 6 North American PEDV strain 1251-125-10.

SEQ ID NO: 16 is the deduced amino acid sequence of the S proteinencoded by SEQ ID NO: 7 Korean PEDV strain SM98.

SEQ ID NO: 17 is the deduced amino acid sequence of the S proteinencoded by SEQ ID NO: 8 Korean attenuated PEDV strain KR-DR13-att.

SEQ ID NO: 18 is the nucleotide sequence of Korean PDCoV strainKOR/KNU14-04/2014 (GenBank Accession No. KM820765).

SEQ ID NO: 19 is the nucleotide sequence of North American PDCoV strainUSA/Iowa459/2014 (GenBank Accession No. KR265865).

SEQ ID NO: 20 is the nucleotide sequence of North American PDCoV strainUSA/Illinois121/2014 (GenBank Accession No. KJ481931).

SEQ ID NO: 21 is the nucleotide sequence of North American PDCoV strainUSA/Minnesota454/2014 (GenBank Accession No. KR265854).

SEQ ID NO: 22 is the nucleotide sequence of North American PDCoV strainUSA/Minnesota455/2014 (GenBank Accession No. KR265855).

SEQ ID NO: 23 is the nucleotide sequence of North American PDCoV strainUSA/Arkensas61/2015 (GenBank Accession No. KR150443).

SEQ ID NO: 24 is the nucleotide sequence of North American PDCoV strainUSA/Minnesota/2013 (GenBank Accession No. KR265853).

SEQ ID NO: 25 is the nucleotide sequence of North American PDCoV strain8734/USA-IA/2014 (GenBank Accession No. KJ567050).

SEQ ID NO: 26 is the nucleotide sequence of North American PDCoV strainUSA/NorthCarolina452/2014 (GenBank Accession No. KR265858).

SEQ ID NO: 27 is the nucleotide sequence of North American PDCoV strainHKU15 MI6148 (GenBank Accession No. KJ620016).

SEQ ID NO: 28 is the nucleotide sequence of North American PDCoV strainHKU15 OH11846 (GenBank Accession No. KT381613).

SEQ ID NO: 29 is the nucleotide sequence of North American PDCoV strainUSA/Illinois272/2014 (GenBank Accession No. KR265856).

SEQ ID NO: 30 is the nucleotide sequence of North American PDCoV strainUSA/Illinois273/2014 (GenBank Accession No. KR265857).

SEQ ID NO: 31 is the nucleotide sequence of North American PDCoV strainHKU15 IL2768 (GenBank Accession No. KJ584355).

SEQ ID NO: 32 is the nucleotide sequence of North American PDCoV strainUSA/Nebraska209/2014 (GenBank Accession No. KR265860).

SEQ ID NO: 33 is the nucleotide sequence of North American PDCoV strainUSA/Nebraska210/2014 (GenBank Accession No. KR265861).

SEQ ID NO: 34 is the nucleotide sequence of North American PDCoV strainHKU15 NE3579 (GenBank Accession No. KJ584359).

SEQ ID NO: 35 is the nucleotide sequence of North American PDCoV strainUSA/Illinois449/2014 (GenBank Accession No. KR265852).

SEQ ID NO: 36 is the nucleotide sequence of North American PDCoV strainUSA/Minnesotal59/2014 (GenBank Accession No. KR265859).

SEQ ID NO: 37 is the nucleotide sequence of North American PDCoV strainUSA/Michigan447/2014 (GenBank Accession No. KR265849).

SEQ ID NO: 38 is the nucleotide sequence of North American PDCoV strainUSA/Michigan448/2014 (GenBank Accession No. KR265850).

SEQ ID NO: 39 is the nucleotide sequence of North American PDCoV strainPDCoV/USA/Iowa136/2015 (GenBank Accession No. KX022602).

SEQ ID NO: 40 is the nucleotide sequence of North American PDCoV strainPDCoV/USA/Nebraska145/2015 (GenBank Accession No. KX022605).

SEQ ID NO: 41 is the nucleotide sequence of North American PDCoV strainPDCoV/USA/Nebraska137/2015 (GenBank Accession No. KX022604).

SEQ ID NO: 42 is the nucleotide sequence of North American PDCoV strainPDCoV/USA/Minnesotal40/2015 (GenBank Accession No. KX022603).

SEQ ID NO: 43 is the nucleotide sequence of North American PDCoV strainHKU15 PA3148 (GenBank Accession No. KJ584358).

SEQ ID NO: 44 is the nucleotide sequence of North American PDCoV strainUSA/Indiana453/2014 (GenBank Accession No. KR265851).

SEQ ID NO: 45 is the nucleotide sequence of North American PDCoV strainUSA/Minnesota292/2014 (GenBank Accession No. KR265864).

SEQ ID NO: 46 is the nucleotide sequence of North American PDCoV strainUSA/Minnesota214/2014 (GenBank Accession No. KR265848).

SEQ ID NO: 47 is the nucleotide sequence of North American PDCoV strainUSA/Minnesota442/2014 (GenBank Accession No. KR265847).

SEQ ID NO: 48 is the nucleotide sequence of North American PDCoV strainHKU15 SD3424 (GenBank Accession No. KJ584356).

SEQ ID NO: 49 is the nucleotide sequence of North American PDCoV strainUSA/Ohio444/2014 (GenBank Accession No. KR265862).

SEQ ID NO: 50 is the nucleotide sequence of North American PDCoV strainUSA/Ohio445/2014 (GenBank Accession No. KR265863).

SEQ ID NO: 51 is the nucleotide sequence of North American PDCoV strainHKU15 KY4813 (GenBank Accession No. KJ584357).

SEQ ID NO: 52 is the nucleotide sequence of Chinese PDCoV isolatePDCoV/CHJXNI2/2015 GenBank Accession No. KR131621).

SEQ ID NO: 53 is the nucleotide sequence of Chinese PDCoV strainCH/SXD1/2015 (GenBank Accession No. KT021234).

SEQ ID NO: 54 is the nucleotide sequence of Chinese PDCoV isolateCHN-JS-2014 (GenBank Accession No. KP757892).

SEQ ID NO: 55 is the nucleotide sequence of Chinese PDCoV isolateCHN-HB-2014 (GenBank Accession No. KP757891).

SEQ ID NO: 56 is the nucleotide sequence of Chinese PDCoV isolateCHN-HN-2014 (GenBank Accession No. KT336560).

SEQ ID NO: 57 is the nucleotide sequence of Chinese PDCoV strain NH(GenBank Accession No. KU981059).

SEQ ID NO: 58 is the nucleotide sequence of Chinese PDCoV strain NHisolate passage 0 (GenBank Accession No. KU981060).

SEQ ID NO: 59 is the nucleotide sequence of Chinese PDCoV strainCH/Sichuan/S27/2012 (GenBank Accession No. KT266822).

SEQ ID NO: 60 is the nucleotide sequence of Chinese PDCoV isolateCHN-AH-2004 (GenBank Accession No. KP757890).

SEQ ID NO: 61 is the nucleotide sequence of Thailand PDCoV strainPDCoV/Swine/Thailand/S5011/2015 (GenBank Accession No. KU051641).

SEQ ID NO: 62 is the nucleotide sequence of Thailand PDCoV strainPDCoV/Swine/Thailand/S5015L/2015 (GenBank Accession No. KU051649).

SEQ ID NO: 63 is the nucleotide sequence of Thailand PDCoV isolateTT_1115 (GenBank Accession No. KU984334).

DETAILED DESCRIPTION I. Definitions

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular forms“a,” “an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. The term “or” refers to a single element ofstated alternative elements or a combination of two or more elements,unless the context clearly indicates otherwise. As used herein,“comprises” means “includes.” Thus, “comprising A or B,” means“including A, B, or A and B,” without excluding additional elements. Allreferences, including patents and patent applications cited herein, areincorporated by reference.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, percentages, temperatures, times, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Accordingly, unless otherwiseindicated, implicitly or explicitly, the numerical parameters set forthare approximations that may depend on the desired properties soughtand/or limits of detection under standard test conditions/methods. Whendirectly and explicitly distinguishing embodiments from discussed priorart, the embodiment numbers are not approximates unless the word “about”is recited.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting.

The terms “administer,” “administering,” “administration,” and the like,as used herein, refer to methods that may be used to enable delivery ofcompositions to the desired site of biological action. These methodsinclude, but are not limited to, intravenous, intramuscular,intradermal, intraperitoneal, subcutaneous, intravaginally, orally,topically, intrathecally, inhalationally, intranasally, transdermally,rectally, and the like. Administration techniques that can be employedwith the agents and methods described herein are found in e.g., Goodmanand Gilman, The Pharmacological Basis of Therapeutics, current ed.;Pergamon; and Remington's, Pharmaceutical Sciences (current edition),Mack Publishing Co., Easton, Pa., which are incorporated herein byreference.

Certain methods of administration deliver the immunogenic composition tomucosal membranes. These include, but are not limited to, intranasal,oral, intravaginal, and rectal. In some embodiments, an adjuvant isselected to facilitate administration to mucosal membranes, and/orstimulate a mucosal immune response. The adjuvant may adhere to themucosal membrane. Mucosal immune responses typically comprise theproduction of IgA antibodies but may also stimulate IgG responses, whichmay be advantageous in certain disclosed embodiments.

Intranasal formulations may include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Non-adjuvant diluents such as water, aqueous saline or otherknown substances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspension, solutions, emulsions, syrups or elixirs, or may bepresented dry in tablet form or a product for reconstitution with wateror other suitable vehicle before use. Such liquid preparations maycontain conventional additives such as suspending agents, emulsifyingagents, non-aqueous vehicles (which may include edible oils), orpreservative.

As used herein, the terms “co-administration,” “administered incombination with,” and their grammatical equivalents, are meant toencompass administration of two or more therapeutic agents to a singlesubject, and are intended to include treatment regimens in which theagents are administered by the same or different routes ofadministration or at the same or different times. In some embodimentsthe one or more compositions described herein will be co-administeredwith other agents, including, but not limited to, therapeutics such asother vaccines, antibiotics, or combinations thereof. These termsencompass administration of two or more agents to the subject so thatboth agents and/or their metabolites are present in the subject at thesame time. They include simultaneous administration in separatecompositions, administration at different times in separatecompositions, and/or administration in a composition in which bothagents are present. Thus, in some embodiments, the compositionsdescribed herein and the other agent(s) are administered in a singlecomposition. In some embodiments, the compositions described herein andthe other agent(s) are admixed in the composition.

The term “effective amount” or “therapeutically effective amount” refersto the amount of an active agent (such as one or more compounds providedherein alone, in combination, or potentially in combination with othertherapeutic agent(s)) sufficient to induce a desired biological result.That result may be amelioration or alleviation of the signs, symptoms,or causes of a disease, or any other desired alteration of a biologicalsystem. The term “therapeutically effective amount” is used herein todenote any amount of a therapeutic and/or preventative that causes animprovement in a disease condition. The amount can vary with thecondition being treated, the stage of advancement of the condition, andthe type and concentration of formulation applied. Appropriate amountsin any given instance will be readily apparent to those of ordinaryskill in the art or capable of determination by routine experimentationsuch as vaccination and observation of an antibody response orvaccination followed by a challenge wherein the vaccinated animalsperform better than non-vaccinated animals that are challengedsimilarly.

The compositions provided herein, alone or in combination with othersuitable components, can be made into aerosol formulations (e.g., theycan be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

The disclosed compositions can be formulated for parenteraladministration, such as, for example, by intravenous, intraarterial,intramuscular, intradermal, intraperitoneal, and subcutaneous routes.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry products ready to be dissolved orsuspended in a pharmaceutically acceptable vehicle for injection,suspensions ready for injection, and emulsions. In addition,controlled-release parenteral dosage forms can be prepared. Suitablematerials for such administration include sterile water; salinesolution; glucose solution; aqueous vehicles, such as sodium chlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose, SodiumChloride Injection, Lactated Ringer's Injection; ethyl alcohol,polyethylene glycol, and propylene glycol; non-aqueous vehicles such as,but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil,ethyl oleate, isopropyl myristate, and benzyl benzoate; aqueous andnon-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this disclosure, compositions can be administered,for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically or intrathecally. In an independentembodiment, parenteral administration, oral administration, and/orintravenous administration are the methods of administration. Theformulations of compounds can be presented in unit-dose or multi-dosesealed containers, such as ampules, bottles, and vials.

“Coronavirus protein,” such as PEDV or PDCoV protein, as used herein inreference to disclosed embodiments refers to any polypeptide productencoded by the coronavirus genome and/or produced only as a result ofcoronavirus infection or the coronavirus lifecycle. As used herein, a“polypeptide” has a length of from 2 amino acids to a length of aprotein encoded by the coronavirus or a cell infected by a coronavirus,such as from 6 amino acids to a length of a protein, and/or a lengthsufficient to produce an antigenic response in a subject administeredthe polypeptide. Thus coronavirus specific polypeptides not encoded by ahost cell but expressed by a coronavirus infected cell are within thescope of the term. Endogenous polypeptides encoded by a coronavirusinfected cell, but expressed in the absence of coronavirus infectionand/or lifecycle, are not intended. However, endogenous polypeptidesexpressed only as a consequence of coronavirus infection and/orlifecycle are within the scope of the term. The term also includesalternative forms of the polypeptides due to changes in secondary and/ortertiary structure, such as those resulting from partial or substantialprotein denaturation as a non-limiting example. Thus denatured forms ofthe polypeptides are within the scope of the term.

PEDV protein refers to any polypeptide product encoded by the PEDVgenome and/or produced as only as a result of PEDV infection or the PEDVlifecycle. Thus PEDV specific polypeptides not encoded by or expressedby a PEDV infected cell are within the scope of the term. Endogenouspolypeptides encoded by a PEDV infected cell, but not expressed in theabsence of PEDV infection and/or lifecycle, are not intended. However,endogenous polypeptides expressed only as a consequence of PEDVinfection and/or lifecycle are within the scope of the term. The termalso includes alternative forms of the polypeptides due to changes insecondary and/or tertiary structure, such as those resulting frompartial or substantial protein denaturation as a non-limiting example.Thus denatured forms of the polypeptides are within the scope of theterm.

The term “antigen” refers to a compound, composition, or substance thatcan stimulate the production of antibodies or a T-cell response in ananimal, including compositions that are injected or absorbed into ananimal. An antigen reacts with the products of specific humoral orcellular immunity. For example, PEDV antigen refers to any portion orfragment of a PEDV polypeptide that is recognized by an anti-PEDVantibody. In some cases, the portion or fragment may be a peptide withan attached moiety, such as, but not limited to, a sugar moiety, aphosphate moiety, or a lipid moiety. Alternatively, the portion orfragment may be a peptide without any attached non-peptide moieties.

Coronavirus antigen, such as a PEDV or PDCoV antigen, refers to anyportion or fragment of a coronavirus polypeptide that is recognized byan anti-coronavirus antibody. In some cases, the portion or fragment maybe a peptide with an attached moiety, such as, but not limited to, asugar moiety, a phosphate moiety, or a lipid moiety. Alternatively, theportion or fragment may be a peptide without any attached non-peptidemoieties.

The term “excipient,” as used in this disclosure, is an additive that isused in combination with the coronavirus antigens. An excipient can beused, for example, to dilute an active agent, such as the coronavirusantigens, and/or to modify properties of a pharmaceutical composition.Examples of excipients include, but are not limited to, water, magnesiumstearate, stearic acid, vegetable stearin, sucrose, lactose, starches,hydroxypropyl cellulose, hydoxypropyl methylcellulose, xylitol,sorbitol, maltitol, gelatin, polyethyleneglycol (PEG), phosphatebuffered saline (PBS), carboxy methyl cellulose, vitamin A, vitamin E,vitamin C, retinyl palmitate, selenium, cysteine, methionine, citricacid, methyl paraben, propyl paraben, sugar, silica, talc, magnesiumcarbonate, sodium starch glycolate, tartrazine, aspartame, benzalkoniumchloride, sesame oil, propyl gallate, sodium metabisulphite, lanolin,polyvinylpyrrolidone (PVP), tocopheryl polyethylene glycol 1000succinate (also known as vitamin E TPGS, or TPGS), dipalmitoylphosphatidyl choline (DPPC), trehalose, sodium bicarbonate, glycine,sodium citrate, lactose, saline, phosphate buffered saline or organicbuffers including but not limited to Tris(hydroxymethyl)aminomethane,and metal chelating agents. Metal chelating agents include, but are notlimited to, organic compounds such as the amino acids glutamic acid andhistidine, organic diacids such as malate, and polypeptides such asphytochelatin, biomolecules such as pyochelin, pyoverdine, enterobactinand Dopa, and synthetic chelates such as ethylenediaminetetraacetic acid(EDTA).

An adjuvant refers to an agent that modifies the effect of anotheragent. As used herein, an adjuvant may be added to an immunogeniccomposition, such as a vaccine, to facilitate a beneficial resultobtained by administering the immunogenic composition, such as bymodifying the immune response to increase the amount of antibodiesproduced and/or increasing the length of protection conferred by thevaccine. An adjuvant may also be added to a composition to helpstabilize a formulation of proteins and/or antigens in a vaccinecomposition. In some embodiments, the adjuvant is a non-naturallyoccurring chemical. Typically water by itself is not an adjuvant.Examples of adjuvants include, but are not limited to, inorganiccompounds, such as alum, aluminum hydroxide, aluminum phosphate,aluminum sulfate, or calcium phosphate hydroxide; mineral oil, such asparaffin oil; organic esters such as aryl or aliphatic esters,particularly alkyl esters such as linear alkyl esters having up to atleast 25 carbons, preferably up to 10 carbons; esters of acids or ofalcohols containing a linear alkyl group, such as plant oils, ethyloleate, propylene glycol di-(caprylate/caprate), glyceryltri-(caprylate/caprate) or propylene glycol dioleate; esters of branchedfatty acids or alcohols, in particular isostearic acid esters;oil-in-water emulsions, such as MF59 (U.S. Pat. No. 6,299,884)(containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85, optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.)), and SAF (containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer LI 21, and threonyl-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion), EMULSIGEN®-based adjuvants includingEMULSIGEN®, EMULSIGEN®-D (containing dimethyldioctadecylammonium bromide(DDA)), EMULSIGEN®-BCL (containing a block copolymer immunostimulant)and EMULSIGEN®-P (containing with a proprietary immunostimulant), andEMULSIGEN®-75 (a double adjuvant comprising an oil-in-water adjuvantwith a cross-linked polymer) (Phibro Animal Health Corporation, Omaha,Nebr., USA); saponins, such as Stimulon™ QS-21 (Antigenics, Framingham,Mass.), described in U.S. Pat. No. 5,057,540, ISCOMATRIX (CSL Limited,Parkville, Australia), described in U.S. Pat. No. 5,254,339, andimmunostimulating complexes (ISCOMS); surfactants, e.g., hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammoniumbromide, N,N-dioctadecyl-N′—N-bis(2-hydroxyethyl-propane di-amine),methoxyhexadecyl-glycerol, and pluronic polyols; copolymers, includinglow molecular weight copolymers such as Polygen™ (available from PhibroAnimal Health Corporation, Omaha, Nebr., USA); polanions, e.g., pyran,dextran sulfate, and poly IC; peptides, e.g., muramyl dipeptide, MPL,aimethylglycine, tuftsin, oil emulsions, alum, and mixtures thereof.

Unsaturated carboxylic acid polymers also may be useful as adjuvants forthe disclosed compositions. Such polymers include polymers of acrylic ormethacrylic acid, copolymers of maleic anhydride and alkenyl derivativeand cross-linked acrylic acid polymers, such as polyacrylate orpolyacrylic acid polymers, optionally cross-linked with polyalkenylethers of sugars or polyalcohols (carbomers). In certain embodiments,polymers comprising moieties having from 2 to 10 carbon atoms, moreparticularly 2 to 4 carbon atoms are preferred, e.g. vinyls, allyls andother ethylenically unsaturated groups. The unsaturated radicals may beoptionally substituted, such as with one or more alkyl moietiesincluding methyl. Such polymers include polymers are sold under the namecarbopol (cross-linked with an allyl sucrose or with allylpentaerythritol) including carbopol 934P, carbopol 971P and carbopol974P (available from Lubrizol Corporation, Wickliffe, Ohio)) and thepolymer sold under the name CARBIGEN™ (available from Phibro AnimalHealth Corporation, Omaha, Nebr., USA). Copolymers of maleic anhydrideand alkenyl derivative include the copolymers EMA (Monsanto) that arecopolymers of maleic anhydride and ethylene. In certain embodiments, anadjuvant comprising an unsaturated carboxylic acid polymer, such as anadjuvant comprising carbopol, or CARBIGEN™, are advantageous foradministration to mucus membranes, such as via intranasal, oral, vaginaland rectal routes.

Other exemplary adjuvants include bacterial lipopolysaccharides;synthetic polynucleotides such as oligonucleotides containing a CpGmotif (e.g., U.S. Pat. No. 6,207,646); IC-31 (Intercell AG, Vienna,Austria), described in European Patent Nos. 1 296 713 and 1 326 634; apertussis toxin (PT) or mutant thereof, a cholera toxin or mutantthereof (e.g., U.S. Pat. Nos. 7,285,281, 7,332,174, 7,361,355 and7,384,640); or an E. coli heat-labile toxin (LT) or mutant thereof,particularly LT-K63, LT-R72 (e.g., U.S. Pat. Nos. 6,149,919, 7,115,730and 7,291,588); bacterial products, such as killed bacteria Bordetellapertussis, Mycobacterium bovis, or toxoids; B peptide subunits of E.coli heat labile toxin or cholera toxin (McGhee, J. R., et al., “Onvaccine development,” Sem. Hematol., 30:3-15 (1993)); nonbacterialorganics, such as squalene or thimerosal; delivery systems, such asdetergents (Quil A); cytokines and/or lymphokines, such as interleukins1-a, 1-β, 2, 4, 5, 6, 7, 8 and 10, 12 (see, e.g., U.S. Pat. No.5,723,127), 13, 14, 15, 16, 17 and 18 (and its mutant forms); theinterferons-a, 3 and y; granulocyte-macrophage colony stimulating factor(GM-CSF) (see, e.g., U.S. Pat. No. 5,078,996 and ATCC Accession Number39900); macrophage colony stimulating factor (M-CSF); granulocyte colonystimulating factor (G-CSF); and the tumor necrosis factors a and β;chemokines, such as MCP-1, MIP-Iα, MIP-Iβ, and RANTES; adhesionmolecules, such as a selectin, e.g., L-selectin, P-selectin andE-selectin; mucin-like molecules, such as CD34, GlyCAM-1 and MadCAM-1; amember of the integrin family, such as LFA-1, VLA-1, Mac-1 and p150.95;co-stimulatory molecules, such as CD40 and CD40L; immunoglobulinsuperfamily members, such as PECAM, ICAMs, e.g., ICAM-1, ICAM-2 andICAM-3, CD2 and LFA-3; growth factors including vascular growth factor,nerve growth factor, fibroblast growth factor, epidermal growth factor,B7.2, PDGF, BL-1, and vascular endothelial growth factor; receptormolecules including Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3,TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, andDR6; Caspase (ICE); muramyl peptides, such asN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE); theB peptide subunits of E. coli heat labile toxin or of the cholera toxin;the RIBI adjuvant system (Ribi Inc.); pluronic polyols; Amphigen;Avridine; L121/squalene; D-lactide-polylactide/glycoside; MPL™(3-O-deacylated monophosphoryl lipid A, Corixa, Hamilton, Mont.;described in U.S. Pat. No. 4,912,094); synthetic lipid A analogs;aminoalkyl glucosamine phosphate compounds (AGP), or derivatives oranalogs thereof (Corixa, Hamilton, Mont.; described in U.S. Pat. No.6,113,918), including 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl2-deoxy-4-O-phosphono-3-O—[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-D-glucopyranoside(529, RC529, optionally formulated as an aqueous form (AF) or as astable emulsion (SE)); and combinations, such as Freund's completeadjuvant or Freund's incomplete adjuvant. Alternatively, oradditionally, the proteins and antigens may be the incorporated intoliposomes for use in an immunogenic composition, such as a vaccine, ormay be conjugated to proteins, such as keyhole limpet hemocyanin (KLH)or human serum albumin (HSA) and/or other polymers.

The term “pharmaceutically acceptable carrier” refers to an excipientthat is a carrier or vehicle, such as a suspension aid, solubilizingaid, or aerosolization aid. Remington: The Science and Practice ofPharmacy, The University of the Sciences in Philadelphia, Editor,Lippincott, Williams, & Wilkins, Philadelphia, Pa., 21^(st) Edition(2005), incorporated herein by reference, describes additionalcompositions and formulations suitable for pharmaceutical delivery ofone or more therapeutic compositions and/or pharmaceutical agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids, such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol, or the like as avehicle. In some examples, the pharmaceutically acceptable carrier maybe sterile to be suitable for administration to a subject (for example,by parenteral, intramuscular, or subcutaneous injection). In addition tobiologically-neutral carriers, pharmaceutical compositions to beadministered can contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate.

The term “immune response” refers to a response of a cell of the immunesystem, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, toa stimulus such as an antigen. An immune response can include any cellof the body involved in a host defense response, including for example,an epithelial cell that secretes an interferon or a cytokine. An immuneresponse includes, but is not limited to, an innate immune response orinflammation. As used herein, a protective immune response refers to animmune response that protects a subject from infection (preventsinfection or prevents the development of disease associated withinfection).

The terms “isolated proteins and antigens” and “isolated coronavirusproteins and antigens,” as used herein, refers to proteins and antigensseparated from the culture medium or supernatant. The isolated proteinsand antigens typically comprise cell material, such as cell wallfragments, and proteins and antigens released from the infected cells,such as by a detergent or freeze-thawing. The isolated proteins andantigens may be in a buffer solution.

The term “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with at least 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequenceidentity to a reference sequence over a specified comparison window.Optimal alignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48:443 (1970). An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution.

The term “vaccine” refers to a preparation of immunogenic materialcapable of stimulating an immune response, administered for theprevention, amelioration, or treatment of disease, such as an infectiousdisease. The immunogenic material may include, for example, antigenicproteins, peptides or DNA derived from them. Vaccines may elicitprophylactic (preventative) and/or therapeutic responses. Methods ofadministration vary according to the vaccine, but may includeinoculation, ingestion, inhalation or other forms of administration asdiscussed herein. Inoculations can be delivered by any of a number ofroutes, including parenteral, such as intravenous, subcutaneous,intramuscular, intranasal, oral, vaginal or rectal. Vaccines may beadministered with an adjuvant to boost the immune response.

II. Overview

Coronaviruses are enveloped viruses and have a positive-sensesingle-stranded RNA genome. The coronavirus genome is typically fromabout 25 to 32 kilobases. Substantially all coronaviruses have spike (S)proteins, envelope, membrane, and nucleocapsid proteins that contributeto the overall viral structure.

With respect to porcine coronaviruses, PEDV is a member of the subfamilyCoronavirinae of the genus Alphacoronavirus. It is an enveloped viruspossessing approximately a 28 kb, positive-sense, single stranded RNAgenome. Although first identified in 1971 in England, variant strains ofPEDV emerging since 2010 in China, and since 2013 in North America, havebeen associated with large-scale outbreaks of diarrhea have been moreacute and severe than those associated with the early Europeanoutbreaks. These recent Chinese and North American strains have beenidentified as belonging to Genogroup 2. Typically, the homology betweenstrains in this genotype is very similar. For example, North Americanstrains typically have about 99% homology. However, the North Americanstrains and recent China strains have less similarity to strains fromEurope and Asia that are Genogroup 1. For example, FIG. 9 shows acomparison of the homologies of seven Genogroup 2 strains from NorthAmerica (SEQ ID NOS: 1-6) and China in 2012 (SEQ ID NO: 9). DR13, anattenuated Korean strain of Genogroup 1 (SEQ ID NO: 8), the sequence ofwhich would be known to a person of ordinary skill in the art based onthe disclosure provided by WO 2015/120378, incorporated herein byreference, as the sequence was known; and SM98, a Korean strain ofGenogroup 1 (SEQ ID NO: 7). SM98 has 96.6 to 96.9% homology with theGenogroup 2 strains, but the Genogroup 2 strains are at least 99%, suchas 99.1-99.9%, homologous with each other.

Porcine deltacoronavirus (PDCoV) was identified in several states acrossthe United Sates in 2014. Symptoms of PDCoV infection include waterydiarrhea, vomiting and dehydration. The genomic sequence of PDCoV isabout 25-26 kilobases, excluding the 3′ poly(A) tail. Typically, PDCoVstrains in the U.S. have at least 99% homology with each other, such asat least 99.2%, at least 99.4%, or at least 99.5% homology with otherU.S. strains. U.S. strains also have at least 85% homology with Chinese,Korean, and/or Thai strains or isolates, such as at least 90%, at least95%, or at least 97% homology. FIGS. 10A-10C shows a comparison of thehomologies of 46 strains or isolates from Korea (SEQ ID NO: 18), NorthAmerica (SEQ ID NOS: 19-51), China (SEQ ID NOS: 52-60) and Thailand (SEQID NOS: 61-63). And FIG. 11 shows the relationship between the strainsor isolates of SEQ ID NOS: 18-63 in terms of nucleotide substitutionsper 100 residues.

Disclosed herein are embodiments of a method for making a coronavirusimmunogenic composition, such as a PEDV or PDCoV immunogeniccomposition, comprising incubating coronavirus infected cells for aneffective period of time to result in one or more replicated coronavirusviral particles being released, such as from 24 to 60 hours or from 24to 48 hours, isolating cells infected with coronavirus away fromcell-free coronavirus virus particles to form cells containingcell-associated coronavirus proteins and antigens, separating thecoronavirus proteins and antigens from the isolated cells to form afirst solution comprising isolated coronavirus proteins and antigens,and optionally inactivating viral particles in the first solution toproduce a second solution. Any embodiments of the method may furthercomprise adding an adjuvant to the second solution. The adjuvant may beany adjuvant suitable for use with the coronavirus proteins andantigens. The adjuvant may be selected to stimulate a mucosal antibodyresponse and/or may be selected for intranasal administration and/orintravaginal administration. The adjuvant may adhere to the mucousmembranes, and/or comprise polyacrylic acid. In any of the disclosedembodiments, the immunogenic composition may be a vaccine and/or may beformulated for intranasal administration. Any embodiments of the methodmay comprise extracting coronavirus proteins and antigens, elutingcoronavirus proteins and antigens, a freeze-thaw cycle, or a combinationthereof. In particular embodiments, the coronavirus is PEDV, or PDCoV.

Also disclosed are embodiments of an immunogenic composition comprisinga first antigenic component comprising isolated coronavirus proteinsand/or antigens from a first coronavirus strain. The immunogeniccomposition may comprise an amount of S protein, such as an amountsufficient to produce an immune response in a subject receiving theimmunogenic composition. The S protein may be an intact S protein. Inany of the above embodiments, the immunogenic composition may comprise asecond antigenic component. The second antigenic component may compriseisolated coronavirus proteins and/or antigens from a second coronavirusspecies and/or strain or isolated proteins and/or antigens from a secondpathogen other than a coronavirus. In some embodiments, second pathogenis porcine reproductive and respiratory syndrome virus, and in otherembodiments, the second pathogen is Mycoplasma hyopneumoniae. However,in certain embodiments when the coronavirus is or comprises PEDV, thesecond pathogen is not Mycoplasma hyopneumoniae.

In any of the above embodiments, the immunogenic composition may be avaccine and/or may comprise an adjuvant selected to stimulate anantibody response. The adjuvant may be selected to stimulate a mucosalantibody response and/or adhere to the mucous membranes. The adjuvantmay comprise a polyacrylic acid adjuvant and/or an emulsifiedoil-in-water adjuvant. The adjuvant may comprise an ammonium salt, suchas a tetraalkylammonium salt, and in certain embodiments, the adjuvantcomprises dimethyldioctadecylammonium bromide.

Also disclosed are embodiments of a method of administering to a subjectan effective amount of any embodiment of the immunogenic compositiondisclosed herein. In some embodiments, the subject is less than 7 daysold, such as 5 days old or less, or 2 days old or less. In any of theabove embodiments, administering may comprise administering orally,intramuscularly, or subcutaneously, or it may comprise administeringintranasally. In particular embodiments, the subject is a swine.

In any of the above embodiments where the subject is a swine, the methodmay comprise administering a first immunogenic composition to a sow, andadministering a second immunogenic composition to at least one pigletfarrowed from the sow, the second immunogenic composition, andoptionally the first immunogenic composition, independently being anyembodiment of the immunogenic composition disclosed herein. In anyembodiments, the second immunogenic composition may be administeredintranasally and/or the first immunogenic composition may beadministered intramuscularly.

In any of the above embodiments, the subject may be a pregnant sow, or asow expected to become pregnant subsequent to administration of thefirst immunogenic composition. The first immunogenic composition may beadministered at a time point prior to the sow becoming pregnant suchthat, when pregnant, the sow has a greater immunity to PEDV compared toa pig not administered the immunogenic composition.

Further disclosed are embodiments of a use of any embodiments of theimmunogenic composition disclosed herein in the manufacture of amedicament for administration to a subject, such as a pig.

III. Method of Extracting or Eluting Coronavirus Proteins and/orAntigens

The disclosure is based in part on the availability and knowledge ofcell culture techniques and their use in the propagation of viruses,such as coronaviruses including, but not limited to, PEDV and PDCoV. MJBiologics, Inc., is also the assignee of U.S. Pat. Nos. 7,241,582,7,449,296, 7,776,537 and 8,142,788, all incorporated herein in theirentirety by reference, which provide additional information relating tocell culture techniques and their use in the propagation of the porcinereproductive and respiratory syndrome virus (PRRSV). The disclosure maybe practiced by use of any suitable cell line susceptible to coronavirusinfection and intracellular replication in vitro. Thus the infected cellmay be any that is capable of being productively infected by thecoronavirus. Non-limiting examples include mammalian cell, such asporcine cells, either in vitro or in vivo. One non-limiting example ofcells in vitro is primary cells from a porcine subject that is infectedwith a coronavirus, such as PEDV or PDCoV. Other non-limiting examplesinclude simian cell lines, such as MA-104; VERO cells; BGM cells; MDCKcells and ST cells. In certain embodiments, MARC cells are used.

With respect to PEDV, the disclosure may be practiced by use of anysuitable cell line susceptible to PEDV infection and intracellularreplication in vitro, such as PEDV having at least a 90% sequenceidentity (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) to SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In certainembodiments, the PEDV has at least 90% sequence identity (i.e., 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%,99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. Thus theinfected cell may be any that is capable of being productively infectedby PEDV. Non-limiting examples include porcine cells, either in vitro orin vivo. One non-limiting example of cells in vitro is primary cellsfrom a porcine subject that is infected with PEDV. Other non-limitingexamples are with the use of a simian cell line, such as MA-104; VEROcells; BGM cells; MDCK cells; MARC cells, and ST cells.

With respect to PDCoV, the disclosure may be practiced by use of anysuitable cell line susceptible to PDCoV infection and intracellularreplication in vitro, such as PDCoV having at least a 90% sequenceidentity (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) to oneor more of SEQ ID NOS: 18-63. In certain embodiments, the PDCoV has atleast 90% sequence identity (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,99.9% or 100%) to one or more of SEQ ID NOS: 19-51. Thus the infectedcell may be any that is capable of being productively infected by PDCoV.Non-limiting examples include porcine cells, either in vitro or in vivo.One non-limiting example of cells in vitro is primary cells from aporcine subject that is infected with PDCoV. Other non-limiting examplesare with the use of a simian cell line, such as MA-104; VERO cells; BGMcells; MDCK cells; MARC cells, and ST cells.

Infection of cells with the coronavirus, such as PEDV or PDCoV, may beat any suitable multiplicity of infection (m.o.i.), such as 0.1, 0.5 or1, and infection of all cells in a culture is not required. In somecases, initial infection of some cells in a culture may be followed bysubsequent release of infectious coronavirus that infects non-infectedcells in the culture. The infected cells may still be used in thepractice of the disclosed methods.

After contact and infection with the coronavirus, the virus is allowedto intracellularly reproduce its proteins and antigens, and soreplicate, for a suitable period of time. The suitable period of timemay vary between different coronaviruses, isolates, strains, and/orsubtypes. In some embodiments, post-infection times range from 1 hour toat least 3 days, such as from 6 hours to 3 days, from 12 hours to 60hours, or from 24 hours to 48 hours. Other post-infection times includeabout 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours,and about 66 hours. Coronavirus culture, such as PEDV or PDCoV culture,typically does not go for more than four days, and thus the harvest maybe a late term harvest, such as after 24 hours. This is in contrast toPRRSV, where the proteins and antigens are typically harvested at anearly term harvest at 28-60 hours post infection. A person of ordinaryskill in the art will understand that ‘early term’ and ‘late term’ arevirus dependent. For example a PRRSV culture will typically take 5 daysto finish, whereas a coronavirus culture, such as PEDV or PDCoV culture,will typically finish after 3 days.

A method of assessing coronavirus protein production over time, afterinfection, also is disclosed to determine possible time points forisolation of infected cells and collection of viral proteins and/orantigens from the cells. In some embodiments, the method comprisestaking samples at different time points after infecting the cells withthe virus. The time points may be based on the percentage of cytopathiceffect (CPE %), such as 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%and/or 90%. Sampling may comprise separating the supernatant from thecells, such as by pouring. The cells may be in a suitable culturevessel, such as a T25, T75 or roller bottle. After separation, anextraction buffer is added to the vessel containing the cells.Typically, the amount of buffer added is from about one half to aboutone tenth volume of the supernatant volume, such as from about onequarter to about one eighth, or from about one fifth to about one sixthvolume of the supernatant volume. The vessel is agitated, such as byrocking and/or rolling. The agitation is performed for a time period andat a temperature sufficient to facilitate extraction of the proteins andantigens. The time period may be from greater than 0 to at least 60minutes, such as from 1 minute to 30 minutes, or from 5 minutes to 20minutes. The temperature may be any temperature suitable to facilitatethe extraction, such as from greater than zero to at least 50° C., from10° C. to 40° C., from 20 OC to 30° C., or about 25° C. The temperaturemay be an ambient temperature. The extraction buffer is then separatedand the extracts analyzed by a suitable technique, such as Western blot,to provide data concerning the presence and/or amount of target antigenversus time. An optimal harvest time is selected based on the data.Typically, the optimal harvest time provides the culture time, or rangeof culture times, that provide a desired CPE % and/or desired targetantigen content.

This “time course” assessment after infection may be used to select apost infection time point for the preparation of viral proteins and/orantigens. The assessment is optionally performed for each coronavirusisolate, strain, and/or subtype. The protein and/or antigen yields mayalso be compared using different coronavirus isolates and different daysafter virus inoculation, and optimal conditions for the highestantigenic yields may be determined by comparative testing.

Coronaviruses produces several proteins, including spike protein. Withrespect to PEDV, the spike protein has a molecular weight of about 152kDa based on deduced amino acid sequences. After post-translationalmodifications the protein may have a molecular weight of from 180 kDa toat least 350 kDa, depending, for example, on the amount ofpost-translational glycosylation. The molecular weight may be determinedby any suitable technique, such as, for example Western blot. PEDV spikeprotein has been predicted to comprise two portions—S1 at the N-terminusand S2 at the C-terminus. The spike protein may be a spike proteinencoded by a PEDV having least 90% sequence identity (i.e., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%,99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, or SEQ ID NO: 9. In certain embodiments, the spike proteinis encoded by a PEDV having least 90% identity (i.e., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, 99.9% or 100%) to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. The spike proteinmay have a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In certain embodiments,the spike protein may have a sequence with at least 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequenceidentity to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,SEQ ID NO: 14, or SEQ ID NO: 15. An indication that two peptidesequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution.

Without being bound to a particular theory, the coronavirus proteins andantigens, such as PEDV and PDCoV proteins and antigens, produced by thedisclosed method include spike proteins in multiple glycosylationstates. This is due, in part, to breaking open the infected cells toseparate and release the proteins and antigens substantially beforeviral particles are released into the culture medium. As a result, spikeprotein glycosylation is in progress, rather than being substantiallycompleted, and therefore, the separated spike proteins have a range ofmolecular weights. By administering to a subject an immunogeniccomposition comprising such spike proteins, the subject is exposed tospike proteins having different glycosylation states, and thus producesantibodies to these proteins. This may be advantageous for the subject,compared to a subject that is administered either a composition thatonly includes non-glycosylated spike protein, such as a recombinantprotein generally from prokaryote, or a composition that hassubstantially only fully glycosylated spike protein, such as aconventional killed vaccine that is typically made with fully grownvirus culture material.

In some embodiments, post-infection times are selected to provide anamount of the spike protein in the composition, such as an extract oreluant, sufficient to provide an immune response, such as a protectiveimmune response, in a subject. In certain embodiments, thepost-infection time is selected such that coronavirus viral particlesare present in the culture medium prior to the collecting, extractingand/or eluting process. In some embodiments, the proteins and/orantigens, such as the spike protein, harvested from coronavirus infectedcells, such as PEDV or PDCoV infected cells, may be in greater amountsthan those available from coronavirus particles in the culture medium,such as at least 2×, 3×, 4×, 5× or more than 5× the amount availablefrom coronavirus particles in the culture medium.

In some embodiments, the proteins and/or antigens harvested fromcoronavirus infected cells may be in greater amounts than thoseavailable from coronavirus particles in the culture medium. At some timepoints after infection, the majority, or entirety, of the replicatedcoronavirus proteins may remain associated with the infected cells orare otherwise part of a cell associated viral component (CAVC). Thus themajority or entirety of coronavirus proteins and/or antigens are eitherwithin the infected cells or associated with the cell membrane of theinfected cells. Under such conditions, relatively few, if any,coronavirus particles are present in the extracellular environment. Thepreparation of CAVC from an early time point, such as before theproduction of coronavirus particles and/or the release thereof into theextracellular environment also has the benefit of increased safety inthat few, if any, infectious viral particles are present as acontaminant. In some embodiments, no infectious viral particles arepresent as a contaminant.

However, in other embodiments, it was surprisingly found that harvestingthe proteins and antigens at a time point after the infected cells hadreleased replicated coronavirus viral particles, such as PEDv or PDCoVviral particles, resulted in improved results. This was in contrast toresults obtained with certain other viruses, such as PRRSV, as disclosedin U.S. Pat. Nos. 7,241,582, 7,449,296, 7,776,537, and 8,142,788.

In some embodiments, the method of preparing coronavirus proteins andantigens from coronavirus infected cells comprises providing apopulation of cells infected with coronavirus; isolating the infectedcells away from cell-free coronavirus to form cells containingcell-associated coronavirus proteins and antigens; and extracting oreluting coronavirus proteins and antigens from the isolated cells. Insome disclosed embodiments, the coronavirus is PEDV. In otherembodiments, the coronavirus is PDCoV. The composition may be asolution. The coronavirus may comprise one or more species and/or one ormore strains of coronavirus, such as two or more species and/or strainshaving at least one nucleotide difference between their genomes.

As used herein, the terms “separating” and “separation” refer to, by wayof unlimited examples, breaking open, extracting, eluting, rupturing,lysing, centrifuging, filtering, or a combination thereof, to releasethe proteins and antigens from within the isolated cells. Thecomposition may also comprise cell fragments. In cases wherein there isno cell-free virus present, then isolating the infected cells away fromcell-free coronavirus may comprise isolating the infected cells fromother materials that may interfere with the method, such as the culturemedium used with the cells. The isolation step may be performed by anymeans known in the art, such as by simply pouring off the medium andleaving the cells to be extracted by detergents or freeze/thaw, or useof centrifugation to generate a cell pellet and supernatant. Thesupernatant can then be removed and/or discarded, such as by use of amembrane filtration, to leave the cells. The separation of viralproteins and antigens may be performed by any suitable method, such asextraction, elution and/or freeze-thawing. The extraction step isoptionally performed by re-suspending the cells in a buffer. In someembodiments, the isolation step may be performed by simply pouring offthe medium and leaving the cells on culture devises such as flasks,roller bottles or cell culture carriers to be extracted by detergents orfreeze/thawing with buffer. The extraction or elution is performed witha detergent-containing solution, thus the buffer used to re-suspendcells may contain detergent. In other embodiments, the extraction may beperformed by freeze-thaw method. Optionally, the viral proteins and/orantigens produced by the method include coronavirus envelope proteins.

In some embodiments, the method comprises using a population of cellsthat has been infected with coronavirus for a sufficient time to producelittle to undetectable amounts of infectious units per ml ofsupernatant, such as the culture media used with the cells. In someembodiments, the time is sufficient to produce tissue culture infectivedoses/ml (TCID₅₀/ml) of from 10¹ to 10¹⁰, such as from 10¹ to 10⁷, orfrom 10¹ to 10⁵⁵. Non-limiting examples include using less than10^(5.5), such as 10⁴ or less, or 10³ or less TCIDso/ml.

The detergent-containing solution may be any that is suitable forextracting or eluting coronavirus proteins and/or antigens. Onenon-limiting example of a suitable class of detergents is non-ionicdetergents. Particular exemplary detergents include, but are not limitedto poly(ethylene glycol) p-isooctyl-phenyl ether,octylphenoxypolyethoxyethanol (Nonidet P-40), or4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100).The detergent is used at a concentration effective for extracting oreluting coronavirus proteins and/or antigens, such as a concentration offrom greater than zero to 5% in solution, such as from greater than zeroto 2%, 0.25% to 1%, or 0.5% in solution. In certain embodiments, thedetergent is a solution of 0.5% Triton X-100. The collecting, extractingand/or eluting may be for from greater than zero to at least about 24hours, such as from 0.1 hours to 24 hours, 0.1 hours to 15 hours, 0.2hours to 10 hours or 0.2 hours to 5 hours, or in certain embodimentsfrom 2 hours to 15 hours. The collecting, extracting and/or, eluting isperformed at a suitable temperature. In some embodiments, thetemperature is from zero to less than 30° C., such as from 0° C. to 25°C., from 1° C. to 20° C., from 2° C. to 10° C. or from 2° C. to 6° C.,and in certain embodiments is 4° C., or room temperature, such as from20 to 25° C. Optionally, the viral proteins and/or antigens produced bythe method include coronavirus envelope proteins.

For a freeze-thaw process a buffer may be added to the cells, typicallyafter the culture medium is removed. The buffer can be any suitablebuffer, such as Tris or phosphate buffered saline (PBS) with or withoutchelating agents, such as EDTA as non-limiting example. The cells arecoated with the buffer, such as by agitation, for example, swirling orstirring. The cells are then placed in a freezer at a temperaturesuitable to freeze the cells and buffer, such as −10° C. or below. Afterthe cells are frozen, they are allowed to thaw. The thawing causes cellsto break, thereby releasing the proteins and antigens. Additionalfreeze-thaw cycles may be performed to release additional proteins andantigens.

After the release of the proteins and antigens, an inactivating agentmay be added. Suitable inactivating agents include any agent that willinactivate viral particles present in the solution of viral protein andantigens, such as binary ethyleneimine (BEI), formalin, or betapropiolactone (BPL). The concentration of activating agent may be from0.01 mol/L to at least 2 mol/L, such as from 0.01 mol/L to 1 mol/L. Thesolution of viral protein and antigens is mixed with the inactivatingagent until inactivation is complete, such as for from 5 minutes to 48hours or more, such as 15 minutes to 2 hours, or from 30 minutes to 1hour. The inactivation process can be stopped by the addition of asufficient amount of thiosulfate solution, such as sodium thiosulfate,to neutralize the excess inactivating agent. After inactivation, thesolution of viral protein and antigens may be optionally diluted beforea suitable adjuvant is added to the solution to produce the immunogeniccomposition.

The immunogenic composition will contain an effective amount ofcoronavirus antigens, such as PEDV or PDCoV antigens, in the solution,the effective amount being readily determined by a person of ordinaryskill in the art. The effective amount may be sufficient to produce adesired immune response in a subject, such as a substantially protectiveimmune response. The amount of coronavirus antigens may typically rangefrom about 1% to about 95% (w/w) of the composition, or even higher orlower if appropriate. The quantity to be administered depends uponfactors such as the age, weight and physical condition of the individualconsidered for vaccination. The quantity also depends upon the capacityof the subject's immune system to synthesize antibodies, and the degreeof protection desired. Effective dosages can be readily established by aperson of ordinary skill in the art through routine trials establishingdose response curves. In some embodiments, the concentration of viralantigens in the solution of coronavirus protein and antigens is from0.01 ng/ml to 10,000 ng/ml, such as from 0.1 ng/ml to 5,000 ng/ml, from0.5 ng/ml to 1,000 ng/ml or from 1 ng/ml to 100 ng/ml. The volume of thedosage is from greater than 0 mL to at least 10 mL, such as from 0.1 mLto 10 mL, from 0.5 mL to 5 mL, or from 0.5 mL to 2 mL.

The coronavirus proteins and antigens may be prepared from coronavirusinfected cells. In certain embodiments, the method comprises preparingthe proteins and antigens from a population of the cells prepared by invitro and in vivo methods. For the in vitro method, VERO cells arecultured, and the cells are harvested following an infection ofcoronavirus.

IV. Compositions and Applications

Disclosed herein are embodiments of a composition comprising theisolated coronavirus proteins and/or antigens prepared according toembodiments of the disclosed method. While a person of ordinary skill inthe art will appreciate that certain disclosed embodiments of the methodconcern isolating proteins and/or antigens from cells and/or culturemedia, the proteins and/or antigens are not necessarily purified. Thus,the composition may further comprise cell fragments, the extractingdetergent, virus-infected cell lysate, or a combination thereof. Thecomposition is suitable for use for any purpose for which coronavirusproteins and/or antigens are used. Non-limiting examples of applicationsof the proteins and/or antigens include the preparation of antibodiesagainst the proteins/antigens; using the proteins and/or antigens asreference markers for coronavirus proteins; and using the proteinsand/or antigens in an immunogenic composition, such as in a vaccineformulation, typically with a suitable adjuvant and optionally with asuitable carrier, and/or excipient. The immunogenic composition may beadministered to a subject, such as an animal, particularly a porcineanime, to generate an immune response. Additional non-limiting examplesof the compositions include those where the protein(s)/antigen(s) is/arein soluble or lyophilized (freeze dried) form.

The disclosed immunogenic composition has a different composition tothat of a conventional coronavirus vaccine, including a conventionalPEDV or PDCoV vaccine, such as a killed or attenuated vaccine.Embodiments of the disclosed method isolate infected cells containingthe coronavirus proteins and antigens away from the supernatant, whichcontains the culture medium, at a time point substantially before viralparticles have been released into the culture medium. Any viral particlethat have been released into the culture medium are removed with thesupernatant. The coronavirus proteins and antigens are then releasedfrom the infected cells by a suitable separation technique, such asextracting and/or eluting, freeze/thawing, or other techniques. In someembodiments, the infected cells may be lysed to release the coronavirusproteins and antigens. Any viral particles also released from theinfected cells are inactivated, such as by a detergent and/or otherinactivating agent. In some embodiments, the detergent is a detergentthat is also used to extract and/or elute the proteins and antigens fromthe infected cells. In other embodiments, the inactivating agent isaffirmatively added to the proteins and antigens. Thus, the compositionmade by the method comprises a high concentration of proteins producedby the infected cells and/or by the virus while within an infected cell,but a low concentration of actual viral particles.

This is in contrast to a killed vaccine, which typically is prepared byallowing the virus to carry the cell infection through the cytopathiceffect (CPE) to substantial release of viral particles. Without beingbound to a particular theory, the proteins produced by the infectedcells and/or by the virus while within an infected cell may be usefulfor production of the viral particles, but are not necessarily properlyexposed to the animal's immune system.

Therefore, the composition and concentration of the proteins andantigens included in the present immunogenic composition is verydifferent from those found in killed or attenuated coronavirus vaccines.

In some embodiments, the immunogenic composition comprises, consistsessentially of, or consists of, coronavirus proteins and antigensprepared by the disclosed method, a buffer solution, an adjuvant, aninactivating agent and a neutralizing agent. In certain embodiments, thebuffer is PBS with EDTA, the inactivating agent is BEI, and/or theneutralizing agent comprises thiosulfate, such as sodium thiosulfate. Inparticular embodiments, the adjuvant is an oil-in-water adjuvant, suchas an EMULSIGEN®-based adjuvant, or an adjuvant that adheres to themucosal membranes, such as an adjuvant comprising polyacrylic acid,typically an adjuvant comprising carbopol such as CARBIGEN™.

In some embodiments, the composition comprises PEDV proteins and/orantigens, and may comprise proteins and/or antigens from one or morestrains of PEDV, such as from 1, 2, 3, 4, 5, 6, or more strains of PEDV.In some embodiments, the composition comprises PDCoV proteins and/orantigens, and may comprise proteins and/or antigens from one or morestrains of PDCoV, such as from 2, 3, 4, 5, 6, or more strains of PDCoV.The composition may comprise proteins and/or antigens from one or morespecies of coronavirus, such as from 1, 2, 3, 4, 5, 6, or more speciesof coronavirus, and each species of coronavirus in the compositionindependently may comprise 1, 2, 3, 4, 5, 6 or more strains of thatparticular species.

In certain embodiments concerning PEDV, strains suitable for use in thecomposition include any strain of PED virus, such as strains from NorthAmerica, Europe and Asia. In particular embodiments, the PEDV strain isa Genogroup 2 strain, and may be a North American strain. In someembodiments, the disclosed immunogenic composition comprises PEDVproteins and antigens encoded by a PEDV strain having at least 90%identity (i.e., 90%, 91%, 92%, 93%, or 94%, or 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%)to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,or SEQ ID NO: 6. In certain embodiments, the PEDV has at least 99%,99.9% or 99.99% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. Exemplary strains include, butare not limited to, the original US PEDV strain, Colorado 2013 (SEQ IDNO: 1), Iowa/18984/2013 (SEQ ID NO: 2), North Carolina USA/NC/2013/35140(SEQ ID NO: 3), Indiana12.83/2013 (SEQ ID NO: 4), Iowa/2013 (SEQ ID NO:5), 1251-125-10 (SEQ ID NO: 6), SM98 (SEQ ID NO: 7), KR-DR13-att (SEQ IDNO: 8), the INDEL strain, the S2aa-del strain, CV777, Chinese PEDVstrains such as Chinese CH/ZMDZY/11, and AH2012 (SEQ ID NO: 9).

In certain embodiments concerning PDCoV, strains suitable for use in thecomposition include any strain of PDCoV virus, such as strains fromNorth America, Europe and Asia. In particular embodiments, the PDCoVstrain is a North American, Chinese, Korean or Thai strain. In someembodiments, the disclosed immunogenic composition comprises PDCoVproteins and antigens encoded by a PDCoV strain having at least 90%identity (i.e., 90%, 91%, 92%, 93%, or 94%, or 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%)to at least one of SEQ ID NOS: 18-63. In certain embodiments, the PDCoVhas at least 99%, 99.9% or 99.99% identity to at least one of SEQ IDNOS: 19-51.

The composition may comprise proteins and/or antigens from at least oneadditional pathogen. The additional pathogen may be any pathogen thatcauses illness and/or an infection in a porcine subject. Exemplarypathogens include, but are not limited to, porcine reproductive andrespiratory syndrome virus (PRRSV), Mycoplasma hyopneumoniae, Mycoplasmahyosynoviae, Mycoplasma rhinitis, Clostridium tetani, Clostridiumperfringens, porcine parvovirus, Erysipelothrix rhusiopathiae,Leptospira pomona, Leptospira grippotyphosa, Leptospira hardjo,Leptospira canicola, Leptospira icterohaemorrhagiae, Leptospirabratislava, porcine circovirus, Lawsonia intracellularis, Escherchiacoli, Actinobacillus pleuropneumoniae, Haemophilus parasuis, Salmonellacholeraesuis, Salmonella typhimurium, Streptococcus suis, Pasteurellamultocida, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae,Serpulina hyodysenteriae, encephalomyocarditis virus, swine influenzavirus, transmissible gastroenteritis virus (TGE), swine deltacoronavirus, rotavirus diarrhea, foot and mouth disease virus, classicalswine fever virus, pseudorabies virus, Japanese encephalitis virus(JEV), encephalomyocarditis virus, or a combination thereof. In certainembodiments, the additional pathogen is not Mycoplasma hyopneumoniae. Inother embodiments, the additional pathogen is Clostridium tetani,Clostridium perfringens, porcine parvovirus, Erysipelothrixrhusiopathiae, Leptospira pomona, Leptospira grippotyphosa, Leptospirahardjo, Leptospira canicola, Leptospira icterohaemorrhagiae, Leptospirabratislava, porcine circovirus, Lawsonia intracellularis, Escherchiacoli, Actinobacillus pleuropneumoniae, Haemophilus parasuis, Salmonellacholeraesuis, Salmonella typhimurium, Streptococcus suis, Pasteurellamultocida, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae,Serpulina hyodysenteriae, encephalomyocarditis virus, swine influenzavirus, transmissible gastroenteritis virus (TGE), rotavirus diarrhea,foot and mouth disease virus, classical swine fever virus, pseudorabiesvirus, Japanese encephalitis virus (JEV), encephalomyocarditis virus, ora combination thereof.

In particular embodiments, the second pathogen is, or comprises, PRRSV.The PRRSV may comprise one or more North American strains, one or moreEuropean strains or combinations thereof. PRRSV strains may include, butare not limited to, Lelystad, VR2332 or HP-PRRSV. In certainembodiments, the PRRSV proteins and/or antigens are extracted or elutedby a method according to one or more of U.S. Pat. Nos. 7,241,582,7,449,296, 7,776,537 or 8,142,788.

In some embodiments, an immunogenic composition as disclosed herein maycomprise coronavirus proteins and/or antigens from one or morecoronavirus species and/or strains, and proteins and/or antigens fromone or more additional pathogens. In certain embodiments, the additionalpathogen comprises PRRSV.

Also disclosed herein are combinations of immunogenic compositions,comprising at least one coronavirus immunogenic composition and at leastone immunogenic composition directed toward a non-coronavirus porcinepathogen. In certain embodiments, the additional pathogen is orcomprises PRRSV. In particular embodiments, the PRRSV immunogeniccomposition is an immunogenic composition prepared according to themethods disclosed in one or more of U.S. Pat. Nos. 7,241,582, 7,449,296,7,776,537 or 8,142,788.

The immunogenic compositions in the combination of immunogeniccompositions may be administered sequentially in any order, or atsubstantially the same time. In some embodiments, the immunogeniccompositions may be mixed to form a single administrable composition. Inother embodiments, the immunogenic compositions are administered asseparate formulations.

The immunogenic compositions disclosed herein may also be administeredin combination with other therapeutic agents suitable for administrationto the subject, such as antibiotics, antiviral agents, antifungalagents, antiparasitic agents, or combinations thereof.

V. Detection of Protective Antibodies Against PEDV

Disclosed herein are embodiments of a method of detecting protectiveantibodies against PEDV. Antibodies against PEDV spike proteins having amolecular weight between 180-kDa to 350-kDa provide immunologicalprotection in livestock such as swine. Accordingly, certain disclosedembodiments provide an agent that binds the antibodies against 180-kDato 350-kDa of porcine epidemic diarrhea virus (PEDV), or PEDV. The agentmay be used in embodiments of a method, device, and/or kit for detectingthe presence of the antibodies against 180-kDa to 350-kDa of PEDV spikeprotein in a biological fluid.

Thus, disclosed herein are embodiments of a method of detectingantibodies against 180-kDa to 350-kDa post translationally modified PEDVspike protein in a sample of a biological fluid from a subject,particularly but not necessarily a porcine subject, suspected of beinginfected with PEDV. The method comprises contacting the sample, or adiluted form thereof, with a binding agent that binds the antibodiesagainst 180-kDa to 350-kDa of PEDV spike protein. The binding of theagent, or agents, to the antibodies against 180-kDa to 350-kDa of PEDVspike protein forms a complex that may be detected to indicate thepresence of the antibodies against 180-kDa to 350-kDa of PEDV spikeprotein post translational variants, and thus the presence of a PEDVinfection in the subject from which the sample was obtained.

The biological fluid may be any fluid in which antibodies against180-kDa to 350-kDa of PEDV spike protein post translational variants maybe present in detectable amounts. Non-limiting examples include bodilysecretions, such as saliva, tears, mucous, nasal discharge, and vaginalsecretions as well as other bodily fluids such as blood, serum, plasma,semen, seminal fluid, milk, and urine as well as any fluid component offeces or a fluid extract of feces.

The binding agent which binds the antibodies against 180-kDa to 350-kDaof PEDV spike protein may be the spike protein, a 180-kDa to 350-kDa ofPEDV spike protein post translational variants, or derivative thereof.In particular, binding agents may also be used to immobilize theantibodies against 180-kDa to 350-kDa of PEDV spike protein, or amacromolecular complex containing it, to facilitate its detection.

Also contemplated are labeled forms of the binding agent to facilitateits detection when bound to antibodies against 180-kDa to 350-kDa ofPEDV spike protein. The binding agent may be labeled to permit directdetection, such as by conjugation to a label which is visible to the eyeupon sufficient aggregation. Alternatively, the binding agent may belabeled for indirect detection, such as by conjugation to an enzyme,which is detected based upon its activity on a detectable substrate orto produce a detectable product. The binding agent may also be unlabeledand then detected based upon use of a detectable reagent, which bindsthe binding agent after formation of the complex. As a non-limitingexample with the use of an antibody as the binding agent, the antibodycomplex comprising antibodies against 180-kDa to 350-kDa of PEDV spikeprotein post translational variants may be detected by a detectablylabeled secondary antibody which binds the antibody bound to theantibodies against 180-kDa to 350-kDa of PEDV spike protein.

In another embodiment, detecting the binding agent complex isfacilitated by immobilization of the complex. In some embodiments, thecomplex is immobilized to a solid substrate comprising an immobilizedsecond binding agent which binds and immobilizes the complex. A firstnon-limiting example of such an embodiment comprises using a secondbinding agent to localize the complexes in discrete areas of thesubstrate to improve detection. In another non-limiting embodiment,immobilization forms a “sandwich” wherein antibodies against 180-kDa to350-kDa of PEDV spike protein are “sandwiched” between the binding agentand a second agent immobilized on a solid substrate which also bindsantibodies against 180-kDa to 350-kDa of PEDV spike protein. As anon-limiting example, the complex may be immobilized by binding to asecond binding agent immobilized to a solid substrate, such as a surfaceof a well, plate, dish or tube. The complex may then by detected basedon localization on the surface. Alternatively the solid substrate may bea bead or chromatographic media which permits detection based onlocalization on the bead or media. The second binding agent preferablybinds the antibodies against 180-kDa to 350-kDa of PEDV spike proteinand the binding agent as described herein. Alternatively, the secondbinding agent is the same as the binding agent.

Also disclosed are embodiments of a device for practicing the abovedescribed method. Generally, such devices are useful for detecting thepresence of antibodies against 180-kDa to 350-kDa of PEDV spike proteinin a sample of a biological fluid as an indicator of PEDV infection inthe subject from which the sample was taken. Thus the devices may beused as a rapid means of diagnosing the presence of PEDV infection.

The test strip may be uniform in composition, such as by being a unitarymembrane strip comprising the first and second portions as describedherein. Non-limiting examples include a strip of nitrocellulose membraneof appropriate pore size. Non-limiting examples of pore sizes includethose in the range of 1-250 microns. Other non-bibulous materials mayalso be used, along with one or more mobilization agent as describedherein to improve the mobilization of a dried first binding agent (thedetector agent or preferably the detector antibody). Non-limitingexamples of a mobilization reagent include glazes comprising sugarand/or BSA (bovine serum albumin).

Alternatively, the test strip is non-unitary in construction but thedifferent components are functionally linked to permit fluidcommunication therebetween. In some embodiments, the first portion ofthe test strip as defined herein is composed of a porous or bibulousmaterial. Non-limiting examples include cellulose or glass wool.

Placement of the first binding agent in a mobilizable form on the firstportion of a device of the invention is preferably by drying a solutioncontaining the agent thereon. In some embodiments, the solution issprayed on and then dried prior to use. A non-limiting representativeexample of such a solution is one containing a detector reagent of theinvention. Preferably, the first binding agent is labeled as describedherein, such as with colloidal gold as a non-limiting example. In otherembodiments, the test strip is within a housing or casing comprisingliquid impermeable material to facilitate the manipulation and use ofthe test strip.

The test strip may be designed to operate solely based on the liquidavailable from a sample applied thereto (see for example U.S. Pat. No.5,591,645 for analogous test strip embodiments). Alternatively, the teststrip may be designed to operate in connection with a solvent ordeveloping solution which increases the volume of the sample applied tothe test strip (see for example U.S. Pat. No. 4,235,601 for analogousembodiments). In other embodiments, the test strip is embodied in ahousing or casing, preferably composed of a plastic, polyacrylate orother liquid resistant material, to form a device of the invention. Thetest strip may include a backing composed of similar materials.

A test strip or other device of the invention may also comprise acontrol site or control region as described herein. The control site orregion may comprise a reagent that produces a color upon being wetted.Non-limiting examples include cobalt chloride, copper chloride, and thelike. Alternatively, the reagent may be a pH indicator which exhibits acolor at the pH of the traversing fluid different from the color in thedry state. In a further alternative, the reagent is one that binds, andthus permits the detection of, a labeled first binding agent regardlessof whether it has bound antibodies against 180-kDa to 350-kDa of PEDVspike protein.

The device may comprise both a first binding agent which bindsantibodies against 180-kDa to 350-kDa of PEDV spike protein to form acomplex and a second binding agent which immobilizes the complex. Thefirst binding agent may thus be viewed as a “detector agent” and is asdescribed herein. Where the first binding agent is 180-kDa to 350-kDa ofPEDV spike protein, it may be viewed as a “detector antigen.” The firstbinding agent may be located in a mobilizable form on a first portion ofthe device. A non-limiting example of how to make such a mobilizablefirst binding agent comprises drying the agent on a first portion of adevice. Upon hydration with a liquid, such as a sample of a biologicalfluid, the agent is mobilized within the sample and thus may move withthe liquid. Where the liquid, such as a sample of a biological fluid,contains antibodies against 200-kDa to 350-kDa of PEDV spike protein,the first binding agent binds the antibodies against 180-kDa to 350-kDaof PEDV spike protein to form a complex which moves with the liquid.

A second binding agent is immobilized on a second portion of a device tobind and immobilize a complex comprising the first binding agent andantibodies against 180-kDa to 350-kDa of PEDV spike protein when such acomplex contacts the second binding agent. The second binding agent maythus be viewed as the “capture agent,” or in the case of an antigen asthe second binding agent, a “capture antigen.” Contact between thesecond binding agent and the complex occurs via the movement of a liquidcontaining the complex, such as a sample of a biological fluid thatcontains a complex of antibodies against 180-kDa to 350-kDa of PEDVspike protein and mobilized first binding agent as described above, intocontact with the second binding agent. Such movement is readilyaccomplished by the first and second portions of the device being influid communication with each other such that fluid in the first portionwill move into and through the second portion. Such fluid communicationmay be direct, with no intervening material between the first and secondportions, or indirect, with an intervening material between the firstand second portions that permits liquid to pass from the first to secondportions.

Detection of immobilized complex in the device, preferably by detectionof a detectably labeled first binding agent immobilized in the secondportion as permitted by the device, may be used to indicate the presenceof antibodies against 180-kDa to 350-kDa of PEDV spike protein in asample of biological fluid. The presence of antibodies against 180-kDato 350-kDa of PEDV spike protein may be used as an indication of PEDVinfection in the subject from which the sample was obtained. The sampleis preferably from a porcine subject, or other subject suspected ofbeing infected with PEDV, but any subject which may be infected by PEDVcarrier may be used in the devices of the invention.

Biological fluids that may be used in the device include any fluid inwhich antibodies against 180-kDa to 350-kDa of PEDV spike protein may bedetectably present. Non-limiting examples have been provided above andbelow, and dilutions of such fluids may also be used as the sample.

The present disclosure provides a binding agent capable of bindingantibodies against 180-kDa to 350-kDa of PEDV spike protein in a sampleof a biological fluid from a subject. Preferably, the binding agentspecifically binds 180-kDa to 350-kDa of PEDV spike protein antibodiesto the exclusion of other molecules present in the biological fluid. Inmany embodiments of the disclosure, the subject is a pig, and thus thesample may be of a bodily fluid or secretion from a pig. Non-limitingexamples of pigs from which samples may be obtained for use with thepresent invention include boar, sow, fattener, gilt, nursery pigs,finishing pigs, and weaned pigs. The pigs may range in age from 1 day toat least 60 days, such as from 1 day to about 30 days, 30 days to about40 days, 41 days to about 50 days, or 51 days to about 60 days or older.

The binding agent preferably, or substantially selectively, binds anantibody against 180-kDa to 350-kDa of PEDV spike protein as found inmultiple PEDV strains and isolates. In other embodiments, the bindingagent does not cross react with other porcine viruses, such ascircovirus, porcine parvovirus (PPV), Japanese encephalitis virus (JEV),rotavirus, pseudorabies, encephalomyocarditis virus, swine influenzavirus, PRRSV or transmissible gastroenteritis (TGE) virus.

The binding agent is preferably a 180-kDa to 350-kDa of PEDV spikeprotein, or a fragment thereof, which binds 180-kDa to 350-kDa of PEDVspike protein antibodies. Accordingly, the disclosure provides animmunochromatographic-based method for detecting PEDV. FIG. 6 provides aWestern blot illustrating detection of antibodies against 180-kDa to350-kDa of PEDV spike protein. With reference to FIG. 6, pigs in cases 1and 2 maintained healthy status without diarrhea, but pigs in cases 3and 4 had severe diarrhea caused by PEDV.

The 180-kDa to 350-kDa of PEDV spike protein may be generated by anyappropriate method known in the art. Suitable methods include, but arenot limited to recombinant, extraction, and/or synthetic methods.

As explained herein, the binding agent may be labeled to facilitate itsdetection, such as, for example, by attachment to another moiety. Themoiety is preferably a detectable label, including a directly detectablelabel, such as a radioactive isotope, a fluorescent label (Cy3 and Cy5as non-limiting examples) or a particulate label. Non-limiting examplesof particulate labels include latex particles, metal sols, and colloidalgold particles. Alternatively, the label may be for indirect detection.Non-limiting examples of labels suitable for indirect detection includean enzyme, such as, but not limited to, luciferase, alkalinephosphatase, and horse radish peroxidase. Other non-limiting examplesinclude a molecule bound by another molecule, such as, but not limitedto, biotin, an affinity peptide, or a purification tag. Preferably, thelabel is covalently attached.

The binding agent may be used to detect antibodies against 180-kDa to350-kDa of PEDV spike protein in a sample of a biological fluid from asubject as described herein. The sample is preferably from an individualsuspected of being infected with PEDV due to the presence of symptomsindicative of an infection. Alternatively, embodiments of the disclosedmethod may be used as part of routine screening of animals, such asthose of a farm to permit rapid identification and isolation of infectedindividuals. Certain embodiments also may be used in specific instances,such as prior to transport or transfer of an animal from one location toanother to permit identification of infection and prevent spread ofinfection.

Also disclosed herein are embodiments of a kit comprising a bindingagent, or a composition and/or device comprising the binding agent, foruse in one or more embodiments of the method disclosed herein. Such kitsoptionally further comprise an identifying description or label orinstructions relating to their use in the methods of the presentinvention. Such a kit may comprise containers, each with one or more ofthe various reagents (typically in concentrated form) or devicesutilized in the methods. A set of instructions will also typically beincluded.

Embodiments of a kit comprising a device may further comprise one ormore additional reagents or pieces of equipment for use with the device.Non-limiting examples of additional materials for inclusion are samplediluent solution, diluent vial, and a dropper for transfer of sample.

IV. Examples Example 1 Preparation of PEDV Proteins and/or Antigens

PEDV proteins may be prepared from a PEDV strain by infectingsusceptible cells in vitro or in vivo, and harvesting the infected cellsat an optimal time to prepare cell-associated viral components. For invitro methods, the antigen(s) may be prepared by a cell culture systemor by using recombinant technologies.

In an exemplary embodiment, MARC cells were pelleted by centrifugation,and the supernatant was removed or discarded. The pellets may beoptionally washed. PEDV proteins and antigens were extracted from thecell pellets by suspending the cell pellets in a 0.05M tris(hydroxymethyl) aminomethane 0.025M EDTA buffer containing 0.5% TritonX-100 at a volume of 5-10 times that of the packed cells. The mixturewas stirred for 2-15 hours at 4° C. and then centrifuged at 10,000 g for1 hour. In another example, the mixture was stirred for 0.5-10 hours at4-25° C. and filtered to remove cell debris.

The resulting supernatant comprised the PEDV proteins and antigens.Optionally, the antigen-containing solution may then undergo one or morefreeze-thawing cycles, one or more of each, followed by an additionalextraction cycle, to further break up intact cells and increase theefficiency of the extraction process. The freeze-thawing process alsomay facilitate ensuring that the antigen solution is non-infectious andallowing its use without a risk of spreading the virus.

FIG. 1 provides a photograph of a Western blot of PEDV proteins. Theproteins were obtained from infected cells by an exemplary embodiment ofthe disclosed method and were mixed with one of two monoclonalantibodies, 6C8 or 3F12, which were selected to detect certain PEDVproteins. Lanes identified as ‘S’ contained pre-stained proteinmolecular weight markers. The other lanes contained samples from a PEDVinfected cell culture medium at the end of culture (lanes 1), an extractof isolated PEDV infected cells diluted 2× (lanes 2), an extract ofisolated PEDV infected cells diluted 3× (lanes 3), an extract ofisolated PEDV infected cells diluted 4× (lanes 4), and an extract ofisolated PEDV infected cells diluted 5× (lanes 5). Surprisingly, in thisexample, both of the monoclonal antibodies used appeared to detectproteins with the same molecular weight.

FIG. 2 provides a photograph of a Western blot of PEDV proteins fromcultures of infected cells over time with a mixture of the twomonoclonal antibodies used in FIG. 1. Lane S contained pre-stainedprotein molecular weight markers. The other lanes contained extractedsamples from isolated PEDV infected cells 24 hours post infection (lane1), 30 hours post infection (lane 2), 35 hours post infection (lane 3),47 hours post infection (lane 4), and 52 hours post infection (lane 5).

The results from these two experiments demonstrated that the disclosedmethod successfully extracted proteins from cells infected with PEDV.Moreover, the Western blots illustrate that the extraction methodresults in a concentrated mixture of proteins compared to a culturemedium, such as the culture medium of a killed virus vaccine. Theconcentration of proteins in the extracts or eluents may be more thantwice the concentration of proteins in the culture medium, such as 4times, 6 times, 8 times, 10 times or more than 10 times theconcentration of proteins in the culture medium.

Example 2 Vaccine Preparation Comprising PEDV Proteins and/or Antigens

A vaccine was prepared using the method described in Example 5 from anexemplary antigen solution prepared by the disclosed method. The vaccinewas administered to sows at 3-5 days pre-farrow in an endemic herd, 150days post clinical break. FIG. 3 shows the data from a test where thevaccinated and non-vaccinated sows were kept in the same room. FIG. 4shows data from a test where vaccinated and non-vaccinated sows werekept in separate rooms. The piglet mortality rates in FIGS. 3 and 4 are15% and 9.6%, respectively, for piglets from the vaccinated sows,compared to 28% and 17.6%, respectively, for piglets from non-vaccinatedsows. FIG. 5 provides baseline data from non-vaccinated herds frommultiple farms, illustrating the pre-wean mortality based on days postinitial whole herd PEDV virus exposure. The data in FIGS. 3-5 clearlyillustrates the efficacy of the exemplary vaccine prepared by thedisclosed method.

Example 3 Preparation of Porcine Deltacoronavirus Proteins and/orAntigens

Porcine deltacoronavirus (PDCoV) proteins and/or antigens may beprepared from a PDCoV strain by infecting susceptible cells in vitro orin vivo, and harvesting the infected cells at an optimal time to preparecell-associated viral components. For in vitro methods, the antigen(s)may be prepared by a cell culture system or by using recombinanttechnologies.

Cells are pelleted by centrifugation, and the supernatant is removed ordiscarded. The pellets may be optionally washed. Porcine deltacoronavirus proteins and antigens are extracted from the cell pellets bysuspending the cell pellets in a buffer, such as 0.05M tris(hydroxymethyl) aminomethane 0.025M EDTA buffer containing 0.5% TritonX-100 at a volume of 5-10 times that of the packed cells. The mixture isstirred for 2-15 hours at 4° C. and then centrifuged at 10,000 g for 1hour. The resulting supernatant comprises the PDCoV proteins andantigens. Optionally, the antigen-containing solution may then undergoone or more freeze-thawing cycles, one or more of each, followed by anadditional extraction cycle, to further break up intact cells andincrease the efficiency of the extraction process. The freeze-thawingprocess also may facilitate ensuring that the antigen solution isnon-infectious and allowing its use without a risk of spreading thevirus.

Example 4

PEDV proteins may be prepared from a PEDV strain by infectingsusceptible cells in vitro or in vivo, and harvesting the infected cellsat an optimal time, typically from 24 to 60 hours post infection, toprepare cell-associated viral components. For in vitro methods, theantigen(s) may be prepared by a cell culture system or by usingrecombinant technologies.

In an exemplary embodiment, the supernatant containing whole virusparticles was poured off from the cells and removed or discarded. Thecells may optionally be washed. PEDV proteins and antigens wereextracted from the cell by suspending the cell pellets in a 0.05M tris(hydroxymethyl) aminomethane 0.025M EDTA buffer containing 0.5% TritonX-100 at one fifth to one eighth volume of culture supernatant volume.The cells were incubated with the buffer for 20 minutes to 1 hour atroom temperature (25° C.) and then optionally frozen. The resultingextract comprised the PEDV proteins and antigens. Optionally, theantigen-containing solution may then undergo one or more freeze-thawingcycles, followed by an additional extraction cycle, to further break upintact cells and increase the efficiency of the extraction process. Thefreeze-thawing process also may facilitate ensuring that the antigensolution is non-infectious and allowing its use without a risk ofspreading the virus. The freeze-thawed solution may be filtered orcentrifuged to remove cell debris. After this extraction process it wasfound that the proteins and antigens were inactivated. Optionally, aninactivating agent such as binary ethyleneimine is added to insureinactivation.

Example 5 Vaccine Preparation

Detergent extracts (DE samples) were prepared from MARC (monkey kidney)cells infected with Colorado (CO), Iowa (IA), and North Carolina (NC),SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively (FIGS. 7-9).FIG. 7 is a Western blot of the detergent extracts. For each extract,2×, 3×, and 4× diluted samples were run (lanes 1-3, respectively). Asample of each full grown virus culture also was run (lane 4).

The three DE samples were mixed in equal volume to make 1,000 mL to forma DE mixture. The DE mixture was inactivated with binary ethyleneimine(BEI) and neutralized with sodium thiosulfate. The resulting solutionhad a volume of about 1,200 mL. Samples taken before and after theinactivation process were tested to confirm that there was no live virusin the DE mixture after the inactivation process. FIG. 8 is a Westernblot of the three DE extracts (lanes 1-3), the DE mixture beforeinactivation (lane 4), the DE mixture after inactivation (lane 5), and amixture of the three viral cultures in equal volume.

The inactivated DE mixture was split into two parts—850 mL (Part A) and350 mL (Part B). Part A was diluted with PBS (650 mL) and mixed with 300mL of Emulsigen-D adjuvant (Phibro Animal Health Corporation, Omaha,Nebr., USA) to form an intramuscular (IM) vaccine (2 mL/dose). Part Bwas not diluted and was mixed with 70 mL of CARBIGEN™ adjuvant (PhibroAnimal Health Corporation, Omaha, Nebr., USA) to form an intranasal (IN)vaccine (1 mL/dose). Each mixture was blended with an industrial gradeblender (Commercial Blender 7011, Model 31BL92, Dynamic Corporation ofAmerica, New Hartford, Conn.). The blended solution was aliquoted into50 mL batches for IM vaccine or 20 mL batches for IN vaccine, and storeduntil use. In trials, the IN vaccine was also used as a subcutaneous(SQ) vaccine.

Example 6 Pre-Wean Mortality in Vaccinated and Unvaccinated Pigs

Table 1 shows multiple farm examples of pre-wean mortality based on dayspost initial whole herd PEDV exposure. The data was used as a baselineto compare vaccine performance.

TABLE 1 Days post Farm Farm Farm Farm Farm Farm All farm exposure 1 2 34 5 6 average 1-7 100 99 97 100 97 100 98%  8-15 98 94 98 98 100 100 98%16-23 51 47 78 89 90 100 76% 24-31 23 24 34 54 45 100 46% 32-39 19 14 2743 34 100 39% 40-47 19 25 17 30 16 22 21% 48-55 24 12 18 27 20 37 23%56-63 19 11 16 25 19 32 20% Individual  15%  14%  15%  20%  17%  12%farm average prior to PEDV

In one trial sows received the PEDV vaccine 3-5 days pre-farrow (endemicinfection, 150 days post clinical break). Treated and non-treatedlitters were in the same room. Table 2 summarizes the results.

TABLE 2 Pigs remaining Piglet Born alive at weaning mortality Vaccinatedsows - 133 (13.3 avg.) 113 (11.3 avg.) 15% 10 head, 2 cc doseNon-vaccinated 405 (12.2 avg.) 291 (8.8 avg.) 28% sows, 33 head

In another trial, sows received the vaccine at 3-5 days pre-farrow (1 ccdose) in an endemic herd (150 days post clinical break) where alllitters within a room were vaccinated or non-vaccinated. The results areshown in Table 3.

TABLE 3 # of Pre-wean litters Born alive Pigs weaned mortality Entireroom 128 1466 (11.4 avg.) 1324 (10.3 avg.) 9.6% vaccinated No vaccine 881059 (12.0 avg.) 872 (9.9 avg.) 17.6% used in room

In another trial, nursing piglets were vaccinated intranasally between2-4 days of age (endemic farm, 150 days post clinical break). Table 4summarizes the results.

TABLE 4 Vaccinated Non- nursing piglets vaccinated (1 cc dose) nursingpiglets # of litters exhibiting 5 of 15 litters (33%) 8 of 27 litters(30%) clinical scour # of litters where 0 of 15 litters (0%)  5 of 27litters (18%) all pigs died Piglet mortality 22% 44% Piglet morbidity12% 13%

In another trial, PEDV vaccine was administered intramuscularly tonursing piglets in an endemic herd (150 days post clinical break). Theresults are shown in Table 5.

TABLE 5 Pigs remaining Pre-wean Born alive at weaning mortality Nursingpiglets 79 (11.2 avg.) 60 (8.6 avg.) 24% 1 cc IM, 2-3 days old, 7litters Nursing piglets 81 (11.5 avg.) 72 (10.3 avg.) 11% 2 cc IM, 2-3days old, 7 litters Nursing piglets 265 (11.0 avg.) 191 (7.9 avg.) 28%no vaccine

In another trial, PEDV vaccine was administered to PEDV naive isoweanpigs at weaning on Day 0. Vaccines were administered subcutaneously(SubQ), intramuscularly (IM), or intranasally (IN). The pigs wereweighed and given a booster vaccine on Day 21. The pigs were challengedand weighed on Day 45, and weighed again on Day 56. The results areshown in Table 6.

TABLE 6 Wean weight Booster Day of and 1^(st) vaccination challengevaccination weight weight End weight Group (Day 0) (Day 21) (Day 45)(Day 56) Controls 12.9 24.9 44.6 54.1 (red tags) SubQ 13.6 23.5 50.861.3 vaccination (green tags) IN vaccination 13.3 27.8 51.0 66.6 (pinktags) IM vaccination 13.4 27.4 48.8 68.0 (purple tags)

Table 7 shows the average daily gain for days 1-45 post vaccination anddays 1-11 post challenge.

TABLE 7 ADG ADG Days 1-45 Days 1-11 Group post vaccination postchallenge Controls 0.71 0.85 (red tags) SubQ vaccination 0.83 0.95(green tags) IN vaccination 0.84 1.41 (pink tags) IM vaccination 0.791.75 (purple tags)

Fecal shedding was evaluated 11 days post challenge in naïve isoweanpigs. The values shown in Table 8 are PCR cycle times (CT) values. Thelower the number, the higher the level of viral material in the sample.The negative cut-off is 35.

TABLE 8 Group Average CT value Range in CT values Controls 28.321.9-33.0 SubQ vaccination 32.8 30.0-33.1 IN vaccination 26.2 21.3-30.0IM vaccination 32.4 31.2-34.7

In another trial, the disclosed vaccine (MJ PEDV) was evaluated againsta commercial PEDV vaccine in an endemic farm 150 days post clinicalbreak. The vaccines were administered 3-5 days pre-farrow. The resultsare summarized in Table 9. The data is a composite of three farrowingsin which all three groups were scattered throughout rooms.

TABLE 9 # of Pigs Pre-wean litters Born alive remaining mortality MJPEDV 36 412 (11.4 avg.) 357 (9.9 avg.) 13.3% 1 cc IM Commercial 50 598(11.9 avg.) 458 (9.1 avg.) 23.4% PEDV 1 cc IM Non-vaccinates 33 422(12.7 avg.) 311 (9.4 avg.) 26.3%

In another trial, the disclosed vaccine (MJ PEDV) was evaluated againsttwo commercial PEDV vaccines during an acute outbreak. Sows farrowed 9to 22 days post PEDV whole herd feedback. The results are shown in Table10.

TABLE 10 Days farrowed Viable pigs post Number of remaining at Group Sowdosage feedback litters in Average 16 days Pre-wean treatment of vaccineexposure group born alive of age mortality Controls No vaccine  9-21days 6 litters 13.1 6.1 53.4% Commercial 2 cc/2 cc 10-22 days 6 litters7.8 4.8 38.4% vaccine 1 Commercial 2 cc/2 cc  9-21 days 7 litters 12.47.2 41.9% vaccine 2 MJ 2 cc/2 cc 12-22 days 10 litters  9.3 6.9 25.8%

Example 7 PEDV Active Farm Trials

A farm experienced an outbreak of PEDV. After feeding back, the herd hadstabilized. A few months after the initial outbreak, bringing in naïvegilts acclimated for PEDV caused a second PEDV event on the farm. Thefarm was farrowing 145-150 sows/week. Each farrowing room included 44crates; each farrowing group occupied 3+farrowing rooms.

Five litters in Room #1 may be selected at 5 days old (day zero). Eachpiglet may be given 1 mL of IN vaccine, and the immunized piglets may bemarked. Mortality may be compared between vaccinated and unvaccinatedlitters at weaning during days 14-19.

Rooms 2-4 may include sows bred at the same time. Sows in Room #2 andhalf the sows in Room #3 may not be vaccinated. The remaining sows inRoom #3 and the sows in Room #4 may be vaccinated with IM vaccine (2cc/sow) on day zero. The sows may farrow on days 14-19. Five litters at5 days old (days 21-26 post sow vaccine) may be selected from Room #4;the selected piglets may be immunized with 1 mL of IN vaccine andmarked. On days 37-44 post sow vaccination, mortality may be compared atweaning among litters that receive no vaccine, litters in which the sowsreceive IM vaccine and the piglets are unvaccinated, and litters inwhich the sows receive IM vaccine and the piglets receive IN vaccine.

Rooms 5-7 may include sows bred at the same time. Sows in Room #5 andhalf the sows in Room #6 may not be vaccinated. The remaining sows inRoom #6 and the sows in Room #7 may be vaccinated with IM vaccine (2cc/sow) on day zero. The vaccinated sows may receive a boostervaccination 14-19 days post initial vaccination. The sows may farrow ondays 28-34 post initial vaccination. Five litters at 5 days old may beselected from Room #7 on days 33-39; the piglets may be vaccinated with1 mL of IN vaccine and marked. On days 47-53, mortality may be comparedat weaning among litters that receive no vaccine, litters in which thesows receive IM vaccine and the piglets are unvaccinated, and litters inwhich the sows receive IM vaccine and the piglets receive IN vaccine.

Example 8 Comparison of Vaccination Protocols

Sows and/or piglets were immunized intramuscularly with 1-4 cc of PEDVvaccine. The piglets were monitored to determine the effect onmortality.

TABLE 11 Room 3 Total Born Pigs Pigs # litters pigs alive wean weanedDose/pig vacci- vacci- per vacci- per Piglet IM nated nated litter natedlitter mortality 1 cc 7 79 11.2 60 8.6 24% 2 cc 7 81 11.5 72 10.3 11.1% — 24 265 11.0 191 7.9 28%

One litter in the 1 cc group of pigs had severe scours. Pigs werevaccinated at 1-2 days of age.

Pigs were weaned at 17-19 days of age.

TABLE 12 Room 4 Pig Pig inventory inventory Dose/pig # sows Born alivefrom vacci- at 15 days Piglet IM vaccinated per litter nated sows of agemortality 2 cc 10 11.2 112 99 11.6% — 33 12.1 399 333 16.7%

Sows were given vaccine between day of farrowing out to day 6pre-farrow. Average timing was 2.2 days pre-farrow for vaccinated sows.

TABLE 13 Room 5 Pig Born inventory Dose/pig # sows alive per from vacci-Pigs Piglet IM vaccinated litter nated sows remaining mortality 4 cc 1013.3 133 113 15%

Example 9 Vaccination of PEDV Naïve Pigs

Forty PEDV naïve isowean pigs (20 days old) may be divided into 4 groupsrandomly and tagged (Groups A, B, C, and D). Four days later, each pigmay be weighted and a blood sample obtained before vaccination. On dayzero, Group A may receive 2 ml of a control vaccine, Group B may receive2 mL of IM vaccine, Group C may receive 1 mL of IN vaccine (0.5 mL×2spots), and Group D may receive 1 mL of SQ vaccine (0.5 mL×2 spots). Onday 21, blood samples may be obtained and each pig may receive a boostervaccination. On day 34, the pigs may be moved to a new location. On day35, a third blood sample may be obtained, and the pigs may be challengedby giving each pig 1 mL of “gut-homogenizer” by mouth. Each pig'sbehavior may be observed daily for two weeks. On day 41 (6 days postchallenge), a fourth blood sample may be obtained. On day 45 (10 dayspost challenge), a fifth blood sample may be obtained.

Example 10 Vaccination of Sows Previously Exposed to PEDV

A PED stabilized farm may be selected. Sero-converting gilts may be keptby the PEDV-feedback method. P₀ sows may be identified in 4 groups—8, 6,4, and 2 prefarrowing groups, 30 gilts per group (15 for control, and 15for vaccination). Blood samples may be obtained before vaccination. Pigsmay be vaccinated with 2 mL of IM vaccine; controls may receivePBS+adjuvant. Blood samples may be obtained before a booster vaccination(2 mL of IM vaccine) at 2, 3, or 4 weeks later.

TABLE 14 Group Day I II III IV 0 B, V B, V B, V B, V 7 14 B, F B, V 21Observe piglet B, V performance (OPP) 28 OPP B, F B, V 35 OPP OPP 42 OPPOPP B, F 49 OPP OPP 56 OPP B, F 63 OPP OPP 70 OPP 77 OPP B = bloodsample, V = vaccination, F = farrowing

Example 11

PEDV proteins usable in vaccines may be prepared from a PEDV strain byinfecting susceptible cells in vitro or in vivo, and harvesting theinfected cells at an optimal time to prepare cell-associated viralcomponents. For in vitro methods, the antigen(s) may be prepared by acell culture system or by using recombinant technologies.

For instance, MARC cells can be grown in cell culture and infected withPEDV either with or without the addition of trypsin. The trypsin isadded at a concentration that will help the virus infect the cells sheetwithout destroying the cells. For instance, at a concentration of 1-10ag/mL. Once the cells show evidence of infection by the PEDV, theculture medium is removed and discarded. The cells may be optionallywashed. A buffer such as Tris or phosphate buffered saline (PBS) withEDTA is added, swirling to coat the cell sheet and then the PEDVproteins and antigens are extracted by placing the culture vessels(roller bottles, flasks, beads or other types of matrix) with the bufferinto a freezer at a temperature at or below −10° C. The vessels areallowed to freeze and then are thawed. Thawing breaks open the cells andreleases the PEDV proteins and antigens useful for preparation of avaccine. Optionally, the thawed culture may be refrozen and rethawed torelease more proteins and antigens and optionally filtered orcentrifuged to remove cell debris. After release of theproteins/antigens, an inactivating agent such as binary ethyleneimine(BEI), formalin, beta propiolactone (BPL) or any other effectiveinactivating agent is added while mixing. Mixing of the inactivatingagent with the culture is continued until inactivation is complete,usually at least 30 minutes. After inactivation, the solution of viralprotein and antigens may be diluted with proper buffer solution like PBSand excipients, and adjuvanted or adjuvanted without further dilution.Acceptable adjuvants include oil-in-water adjuvants such as thosecontaining EMULSIGEN®, adjuvants comprising polymers such as thosecomprising acrylic acids or carbomers such as CARBIGEN™, or other typesof polymers such as POLYGEN™. Once the antigens are inactivated andadjuvanted they may be administered to animals, preferably pigs, viaintramuscular, subcutaneous, intranasal or oral routes.

Example 12

An autogenous immunogenic composition comprising coronavirus proteinsand antigens is produced from a sample received from an infectedsubject. The subject may be a swine. The sample is analyzed to determineif it is infected with a coronavirus, and if so, to determine thespecies and strain of the virus. The virus is then replicated and theproteins and antigens isolated by the method disclosed herein to producean immunogenic composition. Optionally, an adjuvant may be added to thecomposition. An autogenous immunogenic composition produced by thedisclosed method is typically administered to one or more of: (1) theinfected subject; (2) other subjects in an infected population thatincludes or included the infected subject; (3) subjects that may have orhave had contact with the infected subject; or (4) subjects in closeproximity to the infected subject, such as a neighboring building, fieldor farm.

Optionally, one or more coronavirus strains of the same species areidentified and/or selected for the production of a combinationimmunogenic composition. Additionally, or alternatively, isolatedproteins and antigens from one or more additional coronavirus speciesand/or non-coronavirus species may be included in the immunogeniccomposition. The additional strain(s) and/or species may be selected toproduce an improved immune response in a subject when administered incombination with proteins and antigens from the received sample,selected based on prior and/or current infection history of the subjector the subject's location, selected to protect the subject frompotential future infection, or any combination thereof.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method, comprising: isolating coronavirus-infected cellsaway from cell-free coronavirus viral particles in a population of cellsin a culture medium; and extracting or eluting coronavirus proteins andantigens from the isolated coronavirus-infected cells with adetergent-containing solution to form a solution comprising detergentand isolated coronavirus proteins and antigens.
 2. The method of claim1, wherein the coronavirus is a porcine coronavirus.
 3. The method ofclaim 2, wherein the porcine coronavirus is transmissiblegastroenteritis virus, porcine respiratory coronavirus, porcine epidemicdiarrhea virus (PEDV), porcine hemagglutinating encephalomyelitis virus,porcine deltacoronavirus (PDCoV), or a combination thereof.
 4. Themethod of claim 1, further comprising allowing the population of cellsto incubate for a time period sufficient to result in one or morereplicated coronavirus viral particles being released into the culturemedium.
 5. The method of claim 1, wherein the detergent-containingsolution comprises a non-ionic detergent.
 6. The method of claim 5,wherein the non-ionic detergent is poly(ethylene glycol)p-isooctyl-phenyl ether, octylphenoxypolyethoxyethanol (Nonidet P-40),4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100),or a combination thereof.
 7. The method of claim 5, wherein thedetergent-containing solution comprises a detergent at a concentrationof greater than zero to 5% in solution.
 8. The method of claim 5,wherein the detergent-containing solution comprises a chelating agent ata concentration of greater than zero to 0.5M in solution.
 9. The methodof claim 1, wherein extracting or eluting coronavirus proteins andantigens comprises extracting or eluting coronavirus proteins andantigens for about 0.1 to about 15 hours.
 10. The method of claim 9,wherein extracting or eluting coronavirus proteins and antigens isperformed at a temperature of from 0° C. to 25° C.
 11. The method ofclaim 1, wherein the proteins and antigens include coronavirus envelopeproteins.
 12. The method of claim 1, wherein the solution of isolatedcoronavirus proteins and antigens has a concentration of coronavirusproteins and antigens greater than a concentration of coronavirusproteins and antigens in the culture medium that contained thepopulation of infected cells.
 13. The method of claim 1, furthercomprising adding an adjuvant to the solution of isolated coronavirusproteins and antigens.
 14. The method of claim 1, further comprisingadding an excipient to the solution of isolated coronavirus proteins andantigens.
 15. A composition comprising the isolated coronavirus proteinsand antigens from claim 1, and an adjuvant.
 16. The composition of claim15, comprising PEDV proteins and antigens, cell fragments, the detergentand an adjuvant.
 17. The composition of claim 15, comprising PDCoVproteins and antigens, cell fragments, the detergent and an adjuvant.18. A method of vaccinating a subject, the method comprisingadministering to the subject a composition comprising the solution ofisolated coronavirus proteins and antigens from claim 1,
 19. The methodof claim 18, wherein the subject is a porcine subject.
 20. A method ofgenerating an immune response in a porcine subject, the methodcomprising administering to the subject the composition of claim 15.